CN111479932A - Method of treating lymphoma - Google Patents
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- CN111479932A CN111479932A CN201880078523.4A CN201880078523A CN111479932A CN 111479932 A CN111479932 A CN 111479932A CN 201880078523 A CN201880078523 A CN 201880078523A CN 111479932 A CN111479932 A CN 111479932A
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Also disclosed herein are methods of determining the response of a subject having a natural killer cell/T cell lymphoma to pembrolizumab (pembrolizumab) treatment comprising detecting the presence or absence of at least one JAK3 activation mutation or at least one PD-L structural rearrangement.
Description
Cross Reference to Related Applications
This application claims priority to singapore provisional application No. 10201708262R filed on 6.10.2017, the contents of which are incorporated herein by reference in their entirety for all purposes.
Technical Field
The present invention relates generally to the field of molecular biology. In particular, the invention relates to the use of biomarkers for the detection and treatment of cancer.
Background
Natural killer/T cell lymphoma (NKT L) is a rare aggressive malignancy commonly found in asian, mexican and south american populations except japan, it is the most common mature T cell lymphoma in asia.
Studies on several solid tumors, including non-small cell lung, melanoma, and bladder cancers, generally lead to the conclusion that Immunohistochemistry (IHC) PD-L1 positivity is consistent with a greater likelihood of response to PD-1/PD-L blockade, however, PD-L negative tumor patients also have a lower but definite response rate.
Extranodal natural killer/T-cell nasal lymphoma (ENK L) is a rare invasive malignancy commonly seen in asian, mexican and south american populations no targeted therapy has been available to date for the treatment of ENK L since anthracycline-based ENK L regimens have associated poor outcomes, L-asparaginase-based regimens, such as SMI L E (dexamethasone, methotrexate, ifosfamide, L-asparaginase, etoposide) regimens, have significantly improved clinical outcomes, particularly for patients with disseminated disease, however, SMI L E or SMI L E similar regimens still fail in up to 40% to 50% of cases, and the toxicity associated with SMI L E has prevented their use in elderly patients as well.
Furthermore, there is still no FDA-approved targeting regimen to address natural killer/T cell lymphoma (NKT L) because the disease is rare and makes identification of biomarkers responsive to therapy challenging.
Disclosure of Invention
In one aspect, the invention relates to a method of treating a natural killer cell/T cell lymphoma in a subject, the method comprising administering to the subject a therapeutically effective amount of pembrolizumab, wherein the subject is characterized by the presence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement.
In another aspect, the invention relates to a method of determining the response of a subject having a natural killer cell/T cell lymphoma to pembrolizumab treatment, the method comprising obtaining a sample from the subject, detecting the presence or absence of at least one JAK3 activation mutation or at least one PD-L1 structural rearrangement, wherein the presence of at least one JAK activation mutation or at least one PD-L1 structural rearrangement indicates that the subject will respond to pembrolizumab treatment.
In another aspect, the invention relates to a kit for detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement, the kit comprising a detection reagent and at least one pair of primers, wherein the primers are enriched for the genomic region of the JAK3 and PD-L1 genes.
In another aspect, the invention relates to a kit for next generation sequencing that detects the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement.
In another aspect, the invention relates to a kit as disclosed herein for use in a method according to the disclosure herein.
Drawings
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
FIG. 1 shows the genomic profile of 11 pre-treated natural killer/T-cell lymphoma tumors from patients who were subsequently treated with pembrolizumab FIG. 1A shows a ladder of recurrent and mutually exclusive non-silent genomic changes found in 11 pairs of NKT L-normal whole genome sequencing data, the top of the ladder indicates the number of non-silent mutations FIG. 1B shows a schematic of the structural rearrangement of PD-L1 validated in this study FIG. 1C shows positive and lateral scans of positron emission tomography-computed tomography of NKT L1 patients who had obtained complete responses from pembrolizumab before and after treatment with pembrolizumab.
Figure 2 shows a treatment timeline for eleven cases of extranodal natural killer/T-cell lymphoma patients who were administered pembrolizumab after failure of multiple treatment lines.
FIG. 3 refers to somatic mutation genes that recurred in the initial tumors of 11 pembrolizumab-treated patients. The top-to-bottom prioritization of gene ordering is the recurrence and mutual exclusion of genes in the patient cohort that achieved a complete response by pembrolizumab treatment. MAFs from the wAnnovar's 1k genome and the ExAC database of 1% were used as cut-off values.
FIG. 4 shows validation of PD-L1 rearrangements and JAK3 activating mutations identified in natural killer/T cell lymphoma patients fully responsive to pembrolizumab confirmed the breakpoint of each PD-L1 structural rearrangement using Sanger sequencing and JAK3 mutations identified by genome-wide sequencing the gene structure of wild-type (WT) PD-L01 is shown on top for reference the breakpoint involved in each predicted rearranged PD-L1 is shown below white arrows indicate introns and transcriptional orientation, biopsy of all tumors before pembroke administration of pembrolizumab NKT L1, NKT L26, NKT L28 and NKT L31 carry PD-L1 rearrangements, NKT L29 and NKT L30 carry G > A mutations, translation to JAK3 p.a573V.
FIG. 5 shows a schematic representation of tandem replication of the 3' UTR of PD-L1 in NKT L26 deduced from whole genome sequencing data.the wild type region within 9p24.1 has been divided into three blocks (Q, R and S), each shown in a different color.the boundary between Q-R and R-S represents a break point of tandem replication.the rearrangement is heterozygous and the schematic shows that the wild type allele in the normal sample and the wild type and mutant alleles in the tumor are matched.the total copy number of PD-L1 in the tumor is three, the mutant allele has PD-L1 with 3' UTR disrupted.the wild type allele contains Q + R + S and the mutant allele contains Q + R1+ R + S when the genomic region of R1+ R + S of the mutant allele is transcribed, the 3' UTR disrupted PD-L1 and wild type PD-L1 will be transcribed from R1 and R, respectively.
SciClone was proposed to analyze only single nucleotide variations called from a genomic region with copy number 2 without loss of heterozygosity (L OH). therefore, copy number and L OH information were obtained using CANVAS.
Figure 7 shows frequent somatic PD-L1 Structural Rearrangements (SRs) revealed by Whole Genome Sequencing (WGS) data from 32 tumor-normal extranodal natural killer cell/T cell lymphoma samples figure 7A shows a ladder of recurrent mutant genes in an expanded cohort of 32 extranodal natural killer cell/T cell lymphoma untreated samples, mutation types affecting each gene are attached to the bottom of the ladder figure 7B refers to a 3-trace circos representation of the somatic SR detected in a freshly frozen WGS sample, the outermost trace represents the main human chromosome from the hs37 reference genome, the middle trace is a histogram depicting the unique number of samples with SR breakpoints in the corresponding genomic region from the smallest zero (inner trace) to the largest eight (outer trace), the inner traces have black arcs, each arc is a UTR SR. that disrupts the 3' of PD-L1 figure 7C shows a schematic of the structural rearrangements of PD-L1 verified in a cohort of 32 untreated samples.
Figure 8 relates to Sanger validation of PD-L rearrangements identified in 32 untreated NKT L sample cohorts using Sanger sequencing to confirm the breakpoint (indicated by dashed line) of each PD-L structural rearrangement identified by whole genome sequencing the gene structure of wild-type (WT) PD-L is shown on top for reference the chromatogram of Sanger sequenced SR is accompanied by a schematic representation of rearranged PD-L white arrows indicate introns and transcriptional orientation all tumors were biopsies before pembroking administration NKT L carries a rearrangement (complex) with a combination of a 3' UTR deletion and an upstream 73bp reverse intron sequence insertion NKT 6341, NKT L526, NKT L and NKT L31 are samples from pembrolizumab-treatment cohorts NKT, NKT L, NKT L, NKT L, NKT L, NKT L and NKT L are all biopsy samples before tumor treatment administration.
Figure 9 shows an aberrant fusion transcript of PD-L1. panel ' NKT L16 ' shows the genomic and transcriptome structure of PD-L11 translocated to chromosome 6 in sample NKT L016. panel ' NKT L26 ' shows a complex intrachromosomal rearrangement in sample NKT L6 with a 3' UTR deletion accompanied by insertion of an upstream 73bp reverse intron sequence. panel ' NKT L15 ' shows a tandem copy in sample NKT L15. panel ' NKT L4 ' shows an intrachromosomal deletion in sample NKT L4. panel ' NKT L17 ' also shows an intrachromosomal deletion in sample NKT L17. dotted lines and arrows indicate break points and fusion orientation, respectively. Q, R and S blocks represent transcript blocks. triangles represent the orientation of the transcription, while polyadenylation (polyA) signals show black arrows. aberrant and wild type PD-L1 mRNA transcript levels obtained from whole transcriptome sequencing data and light grey and grey copy number of cDNA in tandem copy, show changes for each cDNA sequencing event.
Detailed Description
In recent years, Immune Checkpoint (ICP) inhibitors have shown promising Objective Response Rates (ORR) in the treatment of many malignancies-notably, one result shows an 80% objective response rate using programmed death-1 (PD-1 or CD279) inhibitors in relapsed or refractory (RR) hodgkin lymphoma (H L-currently, clinical studies involving non-small cell lung, melanoma and bladder cancer generally lead to the conclusion that Immunohistochemical (IHC) positivity of programmed death-ligand 1 (PD-L1) is consistent with a greater likelihood of response to PD-1/PD-L1 blockade-interestingly, there is also a lower but definite response rate in PD-L1 negative tumor patients.
The present inventors have identified recurrent genetic alterations in patients with relapsed or refractory natural killer/T-cell lymphoma (RR NKT L) who have achieved a Complete Response (CR) with a programmed cell death 1(PD-1) blocking therapy.
With advances in sequencing technology, the germline genetic susceptibility of recurrent somatic mutations, epigenetic modifications, the DDX3X gene, and the H L a-DPB1 gene that alter the JAK-STAT pathway have been found in natural killer/T-cell lymphoma (NKT L) patients, but none of these studies used Whole Genome Sequencing (WGS) techniques.
Whole genome sequencing of paired Natural killer/T-cell lymphoma (NKT L) tumor-normal samples showed that a somatic breakpoint cluster was present within the programmed cell death ligand 1 (PD-L1/CD 274) gene that recurred highly in 36% (4 out of 11) tumors it was demonstrated that these Structural Rearrangements (SRs) disrupt the 3' untranslated region (UTR) of the PD-L1 gene, resulting in aberrant expression of the PD-L1 chimeric transcript.
In one example, in 11 individuals with relapsed or refractory natural killer/T-cell lymphoma (NKT L) treated with pembrolizumab, a structural rearrangement of the PD-L13 'UTR was found in all four responders, but not in four non-responders without being bound by theory, it is believed that the structural rearrangement of the PD-L13' UTR is associated with a blocked response to PD-1 and a decreased M2-macrophage marker, thereby allowing the use of PD-1 blocking therapy for PD-L1 rearranged natural killer/T-cell lymphoma and, in turn, improving the therapeutic outcome in these patients.
Disclosed herein are methods of treating natural killer cell/T cell lymphoma in a subject, comprising administering to the subject a therapeutically effective amount of pembrolizumab, wherein the molecular genomic profile of the subject is characterized by the presence of at least one PD-L1 structural rearrangement in one example, a method of treating natural killer cell/T cell lymphoma in a subject is disclosed, the method comprising administering to the subject a therapeutically effective amount of pembrolizumab, wherein the subject is characterized by the presence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement.
Thus, in one example, the structural rearrangement disrupts the 3' untranslated region (3 ' UTR) of the PD-L1 gene in another example, the PD-L1 structural rearrangement is a mutation in the PD-L1 gene in yet another example, a mutation in the PD-L1 gene disrupts the 3' UTR of the PD-L1 gene.
In one example, the JAK3 activating mutation is, but is not limited to, any one or more of M511I, A572V, A573V, R657Q, V722I, V674A, L857P, R403H, Q501H, E958K. in another example, the JAK3 activating mutation is a single nucleotide substitution (p.A572V or p.A573V) in the JAK3 Gene (JAK3RefSeq Gene ID: NM-000215). in other words, the JAK3 activating mutation is A572V. the terms "JAK 3 activating" mutation "and" JAK3 mutation "are considered interchangeable.
As used herein, the term "mutation" refers to a permanent change in the nucleotide sequence of the genome of an organism or genetic element. Mutations can be, but are not limited to, insertions, deletions, substitutions, translocations, inversions, micro-inversions, duplications, tandem repeats, breakpoints (mutations), and combinations thereof.
As used herein, the term "structural rearrangement" refers to one or more mutations that result in an overall structural change in the relevant nucleic acid sequence. The "structural rearrangements" span the genomic region and the boundaries of the mutation are called breakpoints. For example, where a breakpoint is present in a gene, a mutation as disclosed herein results in a structural change of the gene. Such structural rearrangements may also refer to changes in the chromosome structure comprising the gene or nucleic acid sequence of interest. Thus, in one example, the mutation is a micro-inversion, translocation, tandem repeat, or breakpoint (mutation), or a combination thereof.
As used herein, the term "inversion" refers to an inversion of a nucleic acid sequence within a particular sequence whereby the sequence is excised and inserted in an opposite orientation to that in which it was previously inserted. In other words, the nucleic acid sequence of interest is inverted end-to-end due to the mutation. The term "microexpression" refers to a nucleic acid sequence of 50-1000bp (base pairs) in length. In one example, the mutation is a slight inversion between 150 and 250bp in length. In one example, the mutation is a slight inversion between 200 and 210bp in length. In another example, the mutation is a slight inversion of about 206bp in length.
As used herein, the term "relapse" or "recurrence" refers to the recurrence of a past disorder (such as, for example, a medical disorder). There are medical conditions known to have a prolonged period of relapse (e.g., malaria). Herein, the term "relapse" refers to a situation in which a pre-existing medical condition (e.g., the presence of a particular disease), which has been treated in the past or is no longer present in the subject, has now relapsed or reappeared in the subject.
As used herein, the term "refractory" refers to a disease or condition that is not responsive to any attempted form of treatment. For example, cancer is considered refractory when it is unresponsive to (or resistant to) cancer therapy. Refractory cancers are also referred to as resistant cancers.
Thus, in one example, the natural killer/T cell lymphoma described herein is a relapsed and/or refractory natural killer/T cell lymphoma. In one example, the natural killer cell/T cell lymphoma is recurrent natural killer cell/T cell lymphoma. In another example, the natural killer cell/T cell lymphoma is a refractory natural killer cell/T cell lymphoma.
Response of relapsed or refractory natural killer/T-cell lymphoma (NKT L) patients to pembrolizumab
Eleven patients with natural killer/T-cell lymphoma from singapore, china and hong kong who were relapsed or refractory (RR) to a L-asparaginase containing chemotherapy regimen, included in the present study (table 1) after a median of two treatment sessions (between 1 and 5 treatment sessions), the median age at diagnosis of these eleven pembrolizumab-treated patients was 42 years (ranging between 27 and 66 years of age) and the median follow-up time since pembrolizumab treatment was eleven months (ranging between 2 and 25 months). sixty-four percent (64%; 7 of 11) achieved Complete Response (CR), while 36% (4 of 11) patients had Progressive Disease (PD). two patients (NKT L and NKT L) remained remitted by pembroglizumab for more than two years, which was considered to be relapsed or refractory natural killer/T-cell lymphoma (NKT L) and the median time of T L) events was sustained for at least 6 months of the rare cases of pembrolizumab (539). this was considered to be rare patients.
Thus, in one example, the subject has not previously responded to SMI L E (dexamethasone, methotrexate, ifosfamide, L-asparaginase and etoposide) therapy.
In patients with natural killer/T-cell lymphoma (NKT L), PD-L1-positive failed to classify (stratify) the response to pembrolizumab
To verify whether PD-L-positivity in natural killer/T-cell lymphoma (NKT L) tumors is able to predict response to pembrolizumab, Immunohistochemistry (IHC) was used to determine PD-L-positivity in all 11 pre-treated NKT 5390 tumors the same pathologist evaluated PD-L-positivity in all tumors in this study to ensure consistency (table 2): PD-L-positive variation in tumor cells in patients achieving complete response and progressive disease, PD-L-positivity in pre-treated tumors of fully responsive patients ranged from 6% to 100% while PD-L-staining intensity in progressive disease patients ranged from 35% to 90%. thus, PD-L-staining intensity failed to distinguish patients achieving complete response from patients with progressive disease and patients with progressive disease, interestingly, NKT 6329 had only 6% of tumor cells positive to PD-L, but had no PD-staining intensity in patients achieving complete response to PD-moderate response to PD-961-positive tumor cells, but had no significant PD-staining in patients who had no clinical evidence of a strong PD-positive PD-staining in patients with no PD-moderate to moderate.
Table 2 membrane PD-L1 immunohistochemical staining grade, PD-L1H-score and PD-L1 positive cells in pretreated NKT L tumors of 11 patients who were subsequently treated with pembrolizumab Immunohistochemical (IHC) staining grade: 0, no, 1+, weak, 2+, moderate, 3+, strong, CR, complete response, PD, progressive disease.
Whole Genome Sequencing (WGS) and analysis of ten patients treated with relapsed/refractory Natural killer/T-cell lymphoma (NKT L) pembrolizumab
To identify genomic biomarkers in natural killer/T-cell lymphoma (NKT L) that responded to PD-1 blocking therapy, genome-wide sequencing of normal paired tumor samples obtained from eleven patients subsequently treated with pembrolizumab was performed.sequencing the natural killer/T-cell lymphoma (NKT L) tumor and whole blood or oral swabs to mean depths of 66.1x and 37.5x, respectively (table 3). somatic mutation calls resulting in mean 1.15 Single Nucleotide Variants (SNV) and microindells/Mb. per sample identified mean 39 (range: 1 to 80) somatic non-silencing protein-encoding variants and found comparable to previous reports on whole exon sequencing of fresh frozen natural/T-cell lymphoma (NKT L0) samples (range: 41 to 42). co-10 genes found a recurrence mutation (figure 3) where only the rearrangement-L structure (NKT 48311) and the complete rearrangement of tumor-T-cell rearrangement of the CT L) construct was achieved in vivo after the initial tomographic scanning of PET 631B 11 (CT) tumor cells, the PET 6326B 9) tumor cells had been identified as a complete displacement in response to the PET 631-bolus (p 9) tumor therapy and the PET 6326 patients with the initial CT transition of the PET 6326-T639T-map 2 tumor cells after the complete displacement of the PET 631-T-mediated tumor-mediated map 7 (p displacement).
Table 3: statistics of whole genome sequencing data in this application.
Thus, in one example, a method of treating natural killer/T-cell lymphoma in a subject is disclosed, the method comprising administering to the subject a therapeutically effective amount of pembrolizumab, wherein the subject is characterized by the presence of at least one JAK3 kinase somatic mutation. In another example, the at least one JAK3 activating mutation is an activating somatic mutation. In another example, there is a JAK3 activating mutation. In another example, the JAK3 activating mutation is p.a 573v.
Thus, in one example, a mutation referred to herein is a micro-inversion, an inversion, a translocation, a tandem repeat, or a breakpoint (mutation). In another example, the mutation is a translocation, tandem repeat (or tandem duplication), or micro-inversion.
In NKT L28 and NKT L31, exon 7 of PD-L1 translocated to 2q24.2 and intron 6 of PD-L1 translocated to 6p12.2 (fig. 4), respectively, in NKT L26, the right breakpoint of tandem replication was located within the 3' UTR of PD-L1 and the left breakpoint was verified to be about 32kbp upstream (fig. 4), this replication event produced a disrupted copy of the 3' UTR and a wild-type copy of PD-L1 (fig. 5), the final PD-L1 structural rearrangement in NKT L1 consisted of 206bp microreliefs located entirely within the 3' UTR of PD-L1 (fig. 4), these somatic changes were not present in the initial tumors of four patients with progressive disease following pembrolizumab treatment.
In addition to sequence analysis by the inventors' genomic tract, visual inspection of the known recurrent mutant genes of natural killer/T-cell lymphoma (NKT L) was also performed to avoid artifacts no mutations in genes associated with antigen presentation and the interferon gamma pathway were found in the cohort analyzed, which are known to be associated with resistance to immune checkpoint blockade in melanoma.
Modulating activity of PD-L13' UTR in Natural killer/T cell lymphoma (NKT L)
To determine the functional significance of this slight inversion in regulating PD-L expression, wild-type and mutant (with 206bp inversion) PD-L ' UTR were cloned into luciferase reporter assay systems and transfected into lymphoma and leukemia cell lines, i.e., NK-S1, K-562 and Jurkat (fig. 10A) results indicate that wild-type PD-L ' UTR can effectively inhibit luciferase activity of reporter proteins, and the identified slight inversion can alleviate this inhibition in NK-S1, K-562 and Jurkat cell lines (P0.01, P0.01 and P03T 03) in the range of PD-3625, K-366313B-T cells (P0.01, P0.03T) to the theoretical extent of tumor killing by the two-fold tumor cells) to confirm that this slight inversion is not mediated by PD-L ' UTR ' or PD-L ' UTR function (fig. 10A) in the regulation of PD-L expression of PD-L, and that the two-fold tumor cells (P-3613) are not directly restricted by the PD-T6313B-T9 + T expression (fig. 3).
Structural rearrangement of PD-L1 and JAK3 activating mutations in natural killer/T cell lymphoma (NKT L) are clonal
Although the response mechanism to PD-L13' UTR structural rearrangement and to the PD-1 blockade of JAK 3-activating mutations remains to be elucidated, it was investigated whether these altered clonality could support the complete response of patients from the single-agent regimen of pembrolizumab, who already had PD-L1 and JAK3 alterations in their pre-treatment tumors from the somatic mononucleotide variation, it was possible to obtain 10 clonal structure solutions (SciClone did not have a clonal solution against NKT L1), five, four complete response cases and one progressive disease case, with a clonal architecture (table 4 and fig. 6). identified somatic PD-L1 and JAK3 mutations were located in their respective pre-treatment tumor establishment clones.
Table 4: genomic-related clonal persistence of pembrolizumab responses in pretreated tumors of the study cohort. CR, complete response; PD, progressive disease.
TABLE 5 PD-L1 and PD-L2 alterations described in hematological malignancies AT LL, adult T cell leukemia/lymphoma, D L BC L, diffuse large B cell lymphoma, H L, Hodgkin's (Hodgkin) lymphoma, NKT L, Natural killer/T cell lymphoma, PCNS L, primary central nervous system lymphoma, PMB L, primary mediastinal B cell lymphoma, PT L, primary testicular lymphoma, UTR, untranslated region.
Results indicate that four of seven NKT L patients (57%) achieve a complete response to PD-1 blocking, have clonal structures with structural rearrangement of the PD-L13 'UTR in their tumors, PD-L13' UTR structural rearrangement has also recently been identified in a single ovarian cancer, where patients achieve a complete response to pembrolizumab, further supporting its role as a potential biomarker of response to PD-1 blocking therapy in natural killer/T cell lymphomas.
Also disclosed herein are methods of determining the response of a subject having a natural killer cell/T cell lymphoma to pembrolizumab treatment, the method comprising obtaining a sample from the subject, detecting the presence or absence of at least one JAK3 activation mutation or at least one PD-L1 structural rearrangement, in another example, the presence of at least one JAK activation mutation or at least one PD-L1 structural rearrangement indicates that the subject will respond to treatment.
As used herein, the term "response" may also be used interchangeably with susceptibility to treatment. The term "susceptibility" refers to the propensity of a substance (e.g., a disease) to be affected by a substance (e.g., a treatment for the disease). This effect may be positive or negative depending on the mentioned features or treatment. For example, if a subject is sensitive to a particular treatment, the subject's susceptibility to the particular treatment is a positive effect. The term "susceptibility" may be interchangeable with, for example, reactivity or sensitivity.
Thus, in one example, the methods disclosed herein are methods of determining the susceptibility of a subject with natural killer/T cell lymphoma to pembrolizumab treatment.
All natural killer/T cell lymphomas are diagnostically EBER + (indicating the presence of epstein-barr virus), and epstein-barr virus (EBV) protein, L MP1, can be considered to constitutively up-regulate PD-L1. without being bound by theory, it is speculated that natural killer/T cell lymphomas will respond to PD-1 inhibitors because they are the native PD-L1 +. indeed, patients with relapsed/refractory natural killer/T cell lymphomas had a primary response to pembrolizumab in previous clinical studies.
In another example, the method comprises administering to the subject an inhibitor selected from the group consisting of a PD-1 inhibitor, a CD279 inhibitor, a PD-L1 inhibitor, a CD274 inhibitor, and combinations thereof.
Also disclosed herein is the use of a compound or inhibitor disclosed herein in the manufacture of a medicament for the treatment of natural killer/T cell lymphoma.
As used herein, the term "inhibitor" refers to a compound that is capable of inhibiting or blocking the activity of a particular receptor or a group of related receptors. The various compounds and drugs are not limited to a single action and may therefore be considered inhibitors of the same receptor, even if they differ structurally and/or chemically. That is, in instances where more than one inhibitor is used, inhibition of a specific receptor is characteristic of these compounds.
Thus, in one example, the inhibitors as disclosed herein are inhibitors that result in blocking the PD-1/PD-L1 axis in another example, the inhibitors are, but are not limited to, PD-1 inhibitors, CD279 inhibitors, PD-L1 inhibitors, CD274 inhibitors, and combinations thereof in yet another example, the methods as disclosed herein comprise administering to a subject an inhibitor that is, but not limited to, a PD-1 inhibitor, a CD279 inhibitor, a PD-L1 inhibitor, a CD274 inhibitor, and combinations thereof.
As used herein, the term "treatment" refers to both prophylactic inhibition of the initial infection or disease as well as therapeutic intervention to alter the natural course of an untreated infection or disease process (e.g., tumor growth or bacterial infection). Treating a disease also refers to a therapeutic intervention that inhibits or suppresses, for example, tumor growth, eliminates a tumor, ameliorates at least one sign or symptom of a disease or pathological condition, or interferes with a pathophysiological process after the disease or pathological condition has begun to develop.
In one example, the treatments or compounds administered to the subject are compounds that block the PD-1/PD-L1 axis in other words, these compounds target immune checkpoints that have an effect on the subject's response to the treatment in one example, these target immune checkpoints are co-inhibitory immune checkpoint molecules in another example, these co-inhibitory immune checkpoint molecules are, but not limited to, CT L a-4, CD80/CD86, PD1, PD-L1/PD-L2, CD80, PD-L1, BT L a, HVEM, TIM3, and GA L9 in another example, the treatments or compounds to be administered to the subject are PD 1/PD-L1 blocking therapies in yet another example, PD 1/PD-L1 blocking therapies are PD-1 blocking therapies.
Thus, in one example, the treatment or compound administered to the subject is a compound that blocks the PD-1/PD-L1 axis in another example, the treatment or compound administered to the subject is a compound that targets PD-1 these compounds can be, but are not limited to, nivolumab (opdivo), pembrolizumab (keyruda), atezolizumab (atezolizumab) (protecntriq), avelizumab (avelumab) (bavancio), daclizumab (durvalumab) (infinzi), pidilizumab (currtmab) (Cure Tech), AMP-224(GlaxoSmithKline), AMP-514 (GlaaxSmithklin), PDR001(Norvartis), cimiciprizumab (semiplicimab) (Regeneron and Sanremolimumab) and combinations thereof.
In addition, within this cohort, PD-L21 immune scores also vary greatly (6%, 2+ to 100%, 3+) and subjects NKT L and NKT L both suffer from progressive disease, although their pre-treated tumors have a high PD-L staining grade, resulting in the occurrence of a single PD-L positive as a biomarker blocking PD-1 in natural killer/T cell lymphomas, as a consequence of the high PD-L staining grade, the problem of using PD-7372 positive alone as a biomarker for PD-1 blocking in natural killer/T cell lymphomas, as a surrogate for the tumor's PD-6342 rearrangement, as a surrogate for other tumor's mechanisms, no rearrangement of PD-635 and JAK3 genes, no alteration of PD-L and JAK3 genes was found in these progressive cases-without the involvement of PD-L and JAK3 genes-without transient blockade of PD-L by initial doses of pembro-bal virus (EBV) 'to be transiently blocked by initial doses of pembrought-1-3-thus, high PD-L positivity not necessarily equates with a good response to PD-1 blockade to PD-1, but with a high PD-7375 staining grade, which is not equivalent to be a surrogate for tumor's tumor-expressing PD-9, as a surrogate for other tumor's, or alternative to be expressed by other mechanisms, as a different in the immunohistochemical tumor' to be expressed by a malignant tumor '8284, as a malignant tumor' alternative to be expressed by a malignant tumor-8284, or similar mechanism.
To determine the prevalence of PD-L and JAK3 alterations, Whole Genome Sequencing (WGS) was performed on 32 more paired tumor-normal natural killer/T-cell lymphoma (NKT L) tumors and corresponding peripheral blood lymphocytes, the clinical pathology information of which is listed in table 6. by mapping the sequencing data to the EBV genome, it was verified that there were no malignant cells in the corresponding peripheral blood of these samples, as the pathogenic virus is known to be present in neoplastic cells, similar to the cohort of 11 pembrolizumab-treated patients, in the extended cohort of the 32 NKT L samples, there was no subsequent pembrolizumab treatment, PD-L was also found to be the most recurrent altered gene in the cohort (fig. 7A), in terms of structural rearrangement, PD-L also appears to be the most rearranged gene (fig. 7B), in these natural killer/T-cell lymphoma (NKT L tumors), the altered forms of PD-L involved in the breakpoint region of PD-5391, in the structural rearrangement region of these natural killer/T cells/T-cells (NKT L tumors), the structural rearrangement was also found using the structural mutation analysis of the early clustering of the accession sites of the accession number of the accession nos. 32, fig. 32, the accession number of the accession sites, 7B-9, the accession number of the accession sites of the accession number of n 7, the accession sites of the accession number of n-accession number of.
Table 6: clinical pathology information of patient
For each PD-L SR, it is possible to identify and validate PD-L1 chimeric transcripts by Sanger sequencing using available Whole Transcriptome Sequencing (WTS) data (fig. 9).
Thus, in one example, a kit for detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement is disclosed, the kit comprising a detection agent and at least one pair of primers in another example, a kit for detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement is disclosed, the kit comprising a detection agent and at least one pair of primers, wherein the primers are enriched for genomic regions of JAK3 and PD-L1 genes.
In another example, the primer pair is, but is not limited to, SEQ ID NO 1 and 2, SEQ ID NO 3 and 4, SEQ ID NO 5 and 6, SEQ ID NO 7 and 8, SEQ ID NO 9 and 10, SEQ ID NO 11 and 12, SEQ ID NO 13 and 14, SEQ ID NO 15 and 16, SEQ ID NO 17 and 18, SEQ ID NO 19 and 20, SEQ ID NO 21 and 22, SEQ ID NO 23 and 24, SEQ ID NO 25 and 26, SEQ ID NO 27 and 28, SEQ ID NO 29 and 30, SEQ ID NO 31 and 32, SEQ ID NO 33 and 34, SEQ ID NO 35 and 36, SEQ ID NO 37 and 38, SEQ ID NO 39 and 40, SEQ ID NO 41 and 42, SEQ ID NO 43 and 44, SEQ ID NO 45 and 3546, and at least one further embodiment of the primers disclosed herein are used to detect the presence of at least one of a mutation in JAK 84, or deletion in a JAK assay kit according to the further examples disclosed herein.
In summary, in the full cohort of 43 natural killer/T-cell lymphoma (NKT L) samples (11 samples were subsequently treated with pembrolizumab and 32 samples were not treated with pembrolizumab), it was shown that frequent (27.9%, 12 out of 43) somatic PD-L13' UTR structural rearrangements in extranodal natural killer/T-cell lymphomas could explain how some extranodal natural killer/T-cell lymphomas escape immune surveillance, thus providing a basis for better treatment of these patients with PD-1 inhibitors.
The presence of recurrent JAK 3-activating mutations in the described cases of complete response is also consistent with reports showing long-term benefit of PD-1 blockade in single lung cancer patients with JAK 3-activating mutations.
Genome characterization of natural killer/T cell lymphomas was shown to correlate with the response of PD-1 blocking therapy using whole genome sequencing data and to indicate that PD-1 blocking therapy can be better selected by genome screening.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and not restrictively. Additionally, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the examples of the invention herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
As used in this application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "genetic marker" includes a variety of genetic markers, including mixtures and combinations thereof.
As used herein, the term "about" in the context of concentration of a formulation component generally refers to +/-5% of the stated value, more generally refers to +/-4% of the stated value, more generally refers to +/-3% of the stated value, more generally refers to +/-2% of the stated value, even more generally refers to +/-1% of the stated value, and even more typically is +/-0.5% of the stated value.
Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the disclosed range. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 3, 4, 5, 6. This is independent of the width of the range.
Certain embodiments may also be broadly and generically described herein. Each of the narrower species and subclass groups belonging to the general disclosure also form part of this disclosure. This includes the generic description of embodiments with a proviso or negative limitation removing any subject matter name from the genus, regardless of whether or not the excised material is specifically recited herein.
The present invention has been described broadly and generically herein. Each of the narrower species and subclass groups falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter name from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. Further, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is thereby also described in terms of any individual member or subgroup of members of the Markush group.
Experimental part
The following examples illustrate methods by which aspects of the invention may be practiced or materials that may be prepared that are suitable for practicing certain embodiments of the invention.
Example 1 materials and methods
Patient and method
Ten patients with relapsed or refractory (RR) natural killer/T-cell lymphoma were treated with pembrolizumab. Responses were assessed by radiation scanning using RECIST criteria. Molecular analysis was performed using Whole Genome Sequencing (WGS) on all prepipimumab tumors and matched normals of eleven patients.
Design of research
For relapsed or refractory (RR) natural killer/T-cell lymphomas, the study cohort consisted of 11 relapsed or refractory (RR) natural killer/T-cell lymphoma patients from singapore, china, and hong kong who were all ineffective based on the L-asparaginase chemotherapy regimen, from previous studies including previously unsequenced NKT L, NKT L, NKT L, NKT L, NKT L, and NKT 201545 from the 2008 world health organization classification, patients were diagnosed with natural killer/T-cell lymphomas with cytotoxic er, CD3+ and EBER + phenotypes were collected 43 out of natural killer/T-cell lymphoma patients with initial sequence and blood/buccal swab samples, wherein 11 patients who failed the L-asparaginase-based chemotherapy regimen were subsequently treated with pembrolizumab for assessment using PET/CT or CT/MRI or EBV combination PCR for assessment of response to all studies using the adept-docks matching study genome (the date of the study review of the study on the study of the euryal-dna-map).
For extranodal natural killer/T-cell lymphoma, all subjects in this study provided written informed consent. Extranodal natural killer/T-cell lymphomas were diagnosed according to the 2008 world health organization classification, with cytotoxic, CD3+ and EBER + phenotypes3. Institutional review boards from SingHealth (2004/407/F), Singapore national university (NUS-IRB-10-250) and Zhongshan university tumor control center (YB2015-015-01) approved the study. Initial tumor and blood samples were collected from 40 extranodal natural killer/T-cell lymphoma patients, six of which were also treated with pembrolizumab following progression to a relapsed or refractory (RR) state. Four of the pembrolizumab-treated patients were from singapore, the remaining two patients were from china. The clinical response of pembrolizumab-treated patients is determined using a combination of signs (e.g., peripheral blood EBV load and PET or CT scan). Of these six patients, freshly frozen tumors were obtained for one patient and formalin-fixed paraffin-embedded (FFPE) tissues were obtained for five patients. WGS data were generated for all 40 tumor-blood samples. Sequencing and alignment statistics can be found in table 7.
Table 7: sequencing and alignment statistics
Genomic DNA extraction
Genomic DNA was extracted from snap-frozen and formalin-fixed paraffin-embedded (FFPE) tumor tissue and whole blood as previously described. The oral swab genomic DNA was purified using the e.z.n.a tissue DNA kit (Omega bio-tek) according to the manufacturer's instructions. Quality and quantity were evaluated as described elsewhere.
NK cell isolation and activation
NK cell isolation peripheral blood mononuclear cells were obtained by centrifugation at 400 × g density for 30 minutes using Ficoll-Paque Plus (GE Healthcare), NK cells were isolated using the EasySep human NK cell isolation kit (STEMCE LL Technologies) NK cells were isolated according to the manufacturer's protocol, NK cell purity was greater than 90% determined by flow cytometry through expression of CD 3-and CD56 +.
The isolated cells were suspended in X-VIVO15 medium (L onza) supplemented with 5% heat-inactivated human serum (Innova Biosciences) with or without 200U/ml I L-2 (Proleukin). 1 × 106The individual cells were seeded in 48-well plates and after 48 hours activation of NK cells was determined by flow cytometry as upregulation of CD25-FITC (clone: M-A251; BD Biosciences) and CD69-BV421 (clone: FN 50; Bio L egend).
NK cells isolation Using human apheresis Cone blood obtained from the health sciences agency of Singapore peripheral blood mononuclear cells were obtained by centrifugation at 400 × g density for 30 minutes using Ficoll-Paque Plus (GE Healthcare), platelets were removed by slow centrifugation at 120 × g for 10 minutes NK cells were isolated using the EasySep human NK cell isolation kit (STEMCE LL Technologies) according to the manufacturer's protocolCell, initial cell concentration 1 × 108Cells/ml.
Isolated NK cells were stained with L ive/DeadaAqua reactive dye (ThermoFisher Scientific) and then surface stained with a monoclonal antibody specific for CD3-V500 (clone: UCHT 1; BD Biosciences) and a monoclonal antibody specific for CD56-PeCy7 (clone: B159; BD Biosciences) to determine the efficiency of isolation, NK cell purity was determined to be greater than 90% by flow cytometry through the expression of CD3-CD56 +.
The isolated cells were resuspended in X-VIVO15 medium (L onza) with or without 200U/ml I L-2 (Proleukin), supplemented with 5% heat-inactivated human serum (Innova Biosciences). 1 × 106The individual cells were seeded in 48-well plates and after 48 hours activation of NK cells was determined by flow cytometry as upregulation of CD25-FITC (clone: M-A251; BD Biosciences) and CD69-BV421 (clone: FN 50; Bio L egend).
Whole genome sequencing
All sequencing libraries were prepared using the TruSeq nano DNA library preparation kit (Illumina) paired end sequencing was performed on either HiSeq2000 or HiSeq X Ten systems (Illumina), 2 × 101bp or 2 × 151 bp. respectively, with a size selection step prior to library preparation of FFPE tumor samples due to high fragmentation of genomic DNA from FFPE material the amplifiable DNA fragment of about 200bp from FFPE samples was used for sequencing library construction to avoid the false negatives believed in the discovery for SR within the PD-L1 gene.
Alternatively, for extranodal natural killer/T cell lymphomas, Whole Genome Sequencing (WGS) was performed on all 40 tumor-normal samples described in this study all sequencing libraries were prepared using the TruSeq Nano DNA library preparation Kit (TruSeq Nano DNA L ibrary Prep Kit) (Illumina.) due to high fragmentation of genomic DNA in FFPE material, a size selection step was performed prior to library preparation of FFPE tumor samples paired end sequencing was performed on HiSeq2000 or HiSeq XTen systems (Illumina) with mean WGS data coverage of 68.9x and 42.2x for 2 × 101bp or 2 × 151 bp. tumors and normal, respectively.
Whole transcriptome sequencing
The sequencing library was prepared using the TruSeqStranded Total RNA library preparation Kit with Ribo-Zero (TruSeq Stranded Total RNA L ibrary Prep Kit) (Illumina) and the Whole Transcriptome Sequencing (WTS) was performed on the HiSeq 2500, HiSeq 3000 or HiSeq X Ten system (Illumina) with read lengths of 2 × 101bp, 2 × 151bp or 2 × 151bp, respectively.
Quantification and normalization of RNA transcripts
RNA reads were aligned to a combined reference of hs37d5 and EBV-1 using STAR in a 2-pass mode. Gene counts were normalized with DESeq2 and the significance of differential expression was calculated using the two-tailed analysis rank sum test. Statistical significance was considered to be p < 0.05.
cDNA Synthesis and real time
Samples with available RNA were reverse transcribed using SuperScript III reverse transcriptase (Invitrogen).
Whole genome and whole transcriptome sequencing
For the purpose of generating WGS data for the extranodal natural killer/T cell lymphoma samples for extranodal natural killer/T cell lymphoma, genomic DNA from snap-frozen and FFPE tumor tissues and whole blood were extracted as previously described. The oral swab genomic DNA was purified using the e.z.n.a tissue DNA kit (Omega Bio-tek) according to the manufacturer's instructions. Quality and quantity were evaluated as described elsewhere. Whole genome sequencing was performed on all tumors and whole blood or buccal swab samples described in this study. All sequencing libraries were prepared using the TruSeq nano DNA library preparation kit (Illumina). FFPE tumor samples were subjected to a size selection step prior to library preparation. RNA extraction and quality and quantity assessment as described previously22. Sequencing libraries were prepared using the TruSeq Stranded Total RNA library preparation kit with Ribo-Zero (Illumina).
Detection and filtration of somatic variations
Sequencing reads were aligned to the hs37d5 reference genome using BWA-MEM. The somatic short variations were detected using Strelka2 and MuSE. Short variants were then annotated by wAnnovar.
Genomic analysis of structural rearrangements
Prior to all downstream analyses gDNA sequencing reads were aligned to the hs37d5 human reference genome using BWA-MEM and repeated by sammbambambambaba labeled PCR (duplicates) in order to identify somatic Structural Rearrangements (SR), man was applied to aligned gDNA reads of tumor-blood paired samples and all predicted SR. within the PD-L1 gene region were validated with Sanger sequencing in order to determine if SR predicted from gDNA sequencing data produced transcripts, cDNA was obtained from the corresponding RNA available using SuperScript III reverse transcriptase (Invitrogen) for PCR-based validation and Sanger sequencing.
Detection and filtration of structural variations
DNA reads were aligned to the hs37d5 reference genome using BWA-MEM and PCR repeats were removed by Sambambambambambaba. Read pairs are marked as inconsistent if they do not align to the reference genome in the expected orientation and/or within the expected insert size. When neither end of the reads matched the reference genome, the reads were marked as clipped.
Manta examined somatic Structural Rearrangements (SRs) and the following filter criteria were followed for each candidate SR: 1) the SR is supported by at least 3 discordant read pairs and at least 3 soft-clip reads, and the sum of all supported reads is at least 10; 2) matching zero-disparity and soft-shear reads present in normal samples; 3) at least 20X coverage in both tumors and matched normal samples; 4) the SR size is at least 1000 bp.
Histograms of unique samples with SR within the genomic region, the SR landscape, were prepared along the main chromosome of hs37d5 in steps of 100kbp with an average sliding window of 1 Mbp. The assumed SR breakpoints are converted to the BEDPE format and visualized as links using CIRCOS with the SR landscape.
Detection of somatic variations
Using FreeBayes6WGS data was analyzed (-X-u-C5-m 30-q 20) and variations with scores less than 30 were filtered out. The single nucleotide variation is predicted to be somatic only when it is called from the tumor rather than matching normal data.
Detection of somatic mononucleotide variations and indels
Somatic single nucleotide variations and indels in WGS data were called using FreeBayes. Candidate variations with scores less than 30 are filtered out. The variant is predicted to be somatic only when the variant is called from a tumor, rather than from matching normal data.
Tumor clonality analysis
The clonal architecture of the tumor was analyzed using SciClone. CANVAS was used to analyze copy number per tumor and loss of heterozygosity information as input for clonality analysis of ScClone.
PCR and Sanger sequencing
The Primer sequences for the discovery cohort of 11 pembrolizumab-treated NKT L patients are listed in table 6 using Primer3 software design primers, the Sanger sequences are aligned to the hs37 reference genome and confirmed with B L AT, optionally, the Primer sequences for the prevalence cohort of 32 NKT L patients who were not subsequently treated with pembrolizumab are also listed in table 7.
TABLE 8 primer pairs for validation of PD-L1 structure rearrangement and JAK3 activation in the discovery cohort of patients subsequently treated with pembrolizumab.
TABLE 9 primer pairs for verifying PD-L1 structural rearrangements in the prevalence cohort for patients subsequently untreated with pembrolizumab.
Histological study and scoring
For recurrent or refractory (RR) natural killer/T-cell lymphomas, PD-L1 IHC analysis was performed with anti-PD-L1 rabbit monoclonal antibody (SP263, Ventana.) PD-L1 positive was assessed as the percentage of tumor cells that stained positively on the cell membrane, or alternatively, PD-L1 expression was assessed as staining at the cell membrane and scored based on the percentage of positive tumor cells and staining intensity for an extra-nodal natural killer/T-cell lymphoma using the following scale 0, no staining, 1+, weak staining, 2+ moderate staining and 3+ strong staining the same pathologist assessed all PD-L1 IHC staining the samples used in this study had available H-scores listed in table 10.
Table 10: available H-score of the sample.
aEvaluated by immunohistochemistry.
bClinical responses reported in Kwong et al, Blood 2017.
Cell lines and constructs
K-562 and Jurkat cell lines were purchased from ATCC, while NK-S1 was generated internally. L GC standard certified K-562 and Jurkat cell lines Jurkat cells were maintained in RPMI 1640(Gibco) supplemented with 10% FBS (HyClone) and K-562 and NK-S1 were grown in DMEM (Gibco) supplemented with 10% FBS (HyClone), 10% horse serum (Gibco) and 2mM L-Glutamine (Gibco). cells were grown at 37 ℃, 5% CO2Grown in the presence and routinely checked for mycoplasma contamination using the mycoaalert mycoplasma detection kit (L onza).
For extranodal natural killer/T cell lymphomas, K-562 and Jurkat cell lines from the ATCC as well as the internal NK-S1 cell line were used to investigate the regulatory role of the minimal structural rearrangements found within the 3' UTR of PD-L1 in a research cohort.
The wild-type PD-L13 ' UTR (ENST00000381573.8) from the SNK6 cell line was cloned into the XhoI and NotI sites of the psiCHECK-2 vector (Promega). for the rearranged partially inverted 3' UTR identified in the summary sample NKT L1, three separate pieces with overhangs were amplified from the wild-type sample (SNK6) and ligated together by PCR.cloning was performed using the Q5 High Fidelity 2X Master Mix (Q5 High-Fidelity 2X Master Mix) (New England Bio L abs.) all cloning primers used to clone the full-length wild-type and mutant PD-L13 ' UTR are described in Table 11.
TABLE 11 cloning primers for cloning the full length 3' UTR of PD-L1 with and without a slight inversion of 206bp length.
Transfection and luciferase assay
For recurrent or refractory (RR) Natural killer/T cell lymphoma, for K-562 and Jurkat, respectively, 5 × 104 Cell sum 6 × 104Cells were seeded in triplicate in 48-well plates and transfected with 250ng plasmid DNA using Liposome 3000Reagent (L ipofectamine 3000Reagent) (Invitrogen.) for NK-S1 cells, 2 × 10 were electroporated in triplicate with 1. mu.g plasmid DNA in 24-well plates using the Neon transfection System (Invitrogen)5The pulse parameters used are as follows, voltage 1300, width 10, number 3 alternatively, for extranodal natural killer/T-cell lymphoma, for K-562 cells, 2.5 × 105Individual cells were seeded in triplicate in 48-well plates and transfected with 250ng plasmid DNA using liposome 3000Reagent (L ipofectamine 3000Reagent) (Invitrogen).For NK-S1 and Jurkat cells, 2.5 × 10 was electroporated in triplicate with 1. mu.g plasmid DNA on 24-well plates using the Neon transfection system (Invitrogen)5A cell.
After 48 hours the cells were lysed with passive lysis buffer (Promega). Luminescence was measured using the dual-luciferase reporter assay system (Promega) and the GloMax-Multi + detection system (Promega). Renilla (Renilla) luciferase activity was divided by firefly luciferase activity and the results were normalized to the empty vector control (mock). Statistical significance was calculated by two-sided t-test. Statistical significance was P < 0.05. All experiments were repeated at least twice.
Data availability
WGS data for 43 natural killer/T-cell lymphoma (NKT L) -normal/blood pairs and whole-transcript sequencing (WTS) data for 28 NKT L were stored in the European Genome Archive (EGA) under the study accession number EGAS 00001002420.
Table 12: additional sequences
Indicates a breakpoint
Sequence listing
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<210>1
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L1-inverted forward primer
<400>1
acataaatac tgtcccgttc ca 22
<210>2
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L1-inverted reverse primer
<400>2
<210>3
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L1-inverted forward primer
<400>3
<210>4
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L1-inverted reverse primer
<400>4
caaccacact cacatgacaa ga 22
<210>5
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L26-replication forward primer
<400>5
cagatacaca tttggaggag acg 23
<210>6
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L26-replication reverse primer
<400>6
<210>7
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L28-translocation forward primer
<400>7
attcaagttt cctttccaga agca 24
<210>8
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L28-translocation reverse primer
<400>8
aagacttttg gttggtattt tctgt 25
<210>9
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L31-translocation forward primer
<400>9
ccatgcacgg tatctcattt aat 23
<210>10
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L31-translocation reverse primer
<400>10
tcagtatctc atcccacctg ac 22
<210>11
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L29-JAK 3-missense forward primer
<400>11
<210>12
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L29-JAK 3-missense reverse primer
<400>12
aagaaaccca cgcatcttct ct 22
<210>13
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L30-JAK 3-missense forward primer
<400>13
<210>14
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L30-JAK 3-missense reverse primer
<400>14
aagaaaccca cgcatcttct ct 22
<210>15
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L27-ARID 1B-insertion Forward primer
<400>15
<210>16
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L27-ARID 1B-insert reverse primer
<400>16
cctcttccta cagtactccc c 21
<210>17
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L4-deletion forward primer
<400>17
caagtttcat tctgtggccc a 21
<210>18
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L4-deletion reverse primer
<400>18
<210>19
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L6-composite forward primer
<400>19
cagatacaca tttggaggag acg 23
<210>20
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L6-composite reverse primer
<400>20
<210>21
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L11-replication forward primer
<400>21
<210>22
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L11-replication reverse primer
<400>22
aactggagtc gaaggtcaca 20
<210>23
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L15-replication forward primer
<400>23
gagaaaacag agggtcaaga agat 24
<210>24
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L15-replication reverse primer
<400>24
gaaaccaaaa gcaagcagga gtag 24
<210>25
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L16-translocation forward primer
<400>25
tccctgacaa ttctaaatcg agt 23
<210>26
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L16-translocation reverse primer
<400>26
<210>27
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L17-deletion forward primer
<400>27
<210>28
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L17-deletion reverse primer
<400>28
caggagaatg gcgtgaactc 20
<210>29
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L35-deletion forward primer
<400>29
ccagaccact tcccatgaaa ttaa 24
<210>30
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L35-deletion reverse primer
<400>30
tactcataca tttggcctca gttg 24
<210>31
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L37-inverted forward primer
<400>31
tataccaaga gatccagtga tggt 24
<210>32
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L37-inverted reverse primer
<400>32
aatcatgttt cagtaccatt ggct 24
<210>33
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L37-inverted forward primer
<400>33
ctactctcca gcccatctat tgag 24
<210>34
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L37-inverted reverse primer
<400>34
atctattgag ggctgatctg gg 22
<210>35
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L4-deletion forward primer
<400>35
acttggtaat tctgggagcc a 21
<210>36
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L4-deletion reverse primer
<400>36
<210>37
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L6-composite forward primer
<400>37
acttggtaat tctgggagcc a 21
<210>38
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L6-composite reverse primer
<400>38
<210>39
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L15-replication forward primer
<400>39
caacacaaca actaatgaga ttttctactg 30
<210>40
<211>26
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L15-replication reverse primer
<400>40
cctcccatga catctctcct cttatg 26
<210>41
<211>26
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L16-translocation forward primer
<400>41
aatgaaagga ctcacttggt aattct 26
<210>42
<211>26
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L16-translocation reverse primer
<400>42
gttttgcagc tagaattcag ttgtaa 26
<210>43
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L17-deletion forward primer
<400>43
cctccaaatg aaaggactca ct 22
<210>44
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L17-deletion reverse primer
<400>44
gcaggtttgg caattctgat tc 22
<210>45
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L35-deletion forward primer
<400>45
cagcattgga acttctgatc ttca 24
<210>46
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L35-deletion reverse primer
<400>46
ggcctcagtt gtcacagtat ttt 23
<210>47
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L37-inverted forward primer
<400>47
tgaaaggact cacttggtaa ttctg 25
<210>48
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L37-inverted reverse primer
<400>48
aggtataata atgctgcctg agat 24
<210>49
<211>32
<212>DNA
<213> Artificial sequence
<220>
<223> CD274-3UTR _ XhoI _ F primer
<400>49
cgtagtctcg agtccagcat tggaacttct ga 32
<210>50
<211>34
<212>DNA
<213> Artificial sequence
<220>
<223> CD274-3UTR _ NotI _ R primer
<400>50
caattagcgg ccgcaacttt ctccactggg atgt 34
<210>51
<211>28
<212>DNA
<213> Artificial sequence
<220>
<223> CD274-3UTR _ F primer
<400>51
tccagcattg gaacttctga tcttcaag 28
<210>52
<211>35
<212>DNA
<213> Artificial sequence
<220>
<223> CD274-A-R primer
<400>52
tgactgagag tctcaaggtc tccctccagg ctccc 35
<210>53
<211>35
<212>DNA
<213> Artificial sequence
<220>
<223> inverted-overhang-F primer
<400>53
cctggaggga gaccttgaga ctctcagtca tgcag 35
<210>54
<211>35
<212>DNA
<213> Artificial sequence
<220>
<223> inverted-overhang-R primer
<400>54
gtcccgttcc aacactgata ctttcaaatg cctga 35
<210>55
<211>35
<212>DNA
<213> Artificial sequence
<220>
<223> CD274-C-F and F2 primers
<400>55
catttgaaag tatcagtgtt ggaacgggac agtat 35
<210>56
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223> CD274-3UTR _ R primer
<400>56
aactttctcc actgggatgt taaactg 27
<210>57
<211>28
<212>DNA
<213> Artificial sequence
<220>
<223> CD274-3UTR _ F primer
<400>57
tccagcattg gaacttctga tcttcaag 28
<210>58
<211>35
<212>DNA
<213> Artificial sequence
<220>
<223> inverted-overhang-R primer
<400>58
gtcccgttcc aacactgata ctttcaaatg cctga 35
<210>59
<211>32
<212>DNA
<213> Artificial sequence
<220>
<223> CD274-3UTR _ XhoI _ F primer
<400>59
cgtagtctcg agtccagcat tggaacttct ga 32
<210>60
<211>34
<212>DNA
<213> Artificial sequence
<220>
<223> CD274-3UTR _ NotI _ R primer
<400>60
caattagcgg ccgcaacttt ctccactggg atgt 34
<210>61
<211>34
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L1 validated Sanger sequences
<220>
<221> misc _ feature
<222>(17)..(18)
<223> breakpoint mutation between reference residues
<400>61
ggcatttgaa agtatcagtg ttggaacggg acag 34
<210>62
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L11 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>62
tgtcatgtga gtgtggttgt gaacagttcc tgaactctga 40
<210>63
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L15 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>63
taagaagaaa gttatattat aatatagttt gcttttacaa 40
<210>64
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L6 (Complex case) 1 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>64
agcgtgacaa gaggaaggaa tgtgccacca tgcccagcta 40
<210>65
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L6 (Complex case) 2 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>65
cgtattggcc aggatagtct agaaaatttt gctaaagcag 40
<210>66
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L4 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>66
tgtgttgtaa agctaagtag ctcaggtact ttgctatccc 40
<210>67
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L17 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>67
catttaagat gagtcagagt tttttgagac ggagtctcgc 40
<210>68
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L16 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>68
caggagaatg ggtatggatg agaacacata cttcctctcc 40
<210>69
<211>41
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L26 validated Sanger sequences
<220>
<221> misc _ feature
<222>(21)..(22)
<223> breakpoint mutation between reference residues
<400>69
ctgatcttca agcaggggat tgatgtgctt tgttaaacag a 41
<210>70
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L28 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>70
atgttaaaag cacgtatttt gaataaaatg ttactttgtc 40
<210>71
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> NKT L31 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>71
ctccctccct ttctctctct ctctctctct ttggtaatgg 40
<210>72
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> F L N375 validated Sanger sequences
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>72
cgtgggatgc aggcaatgtg gaatataaca aataaagcaa 40
<210>73
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> F L N377 validated Sanger sequence
<220>
<221> misc _ feature
<222>(20)..(21)
<223> breakpoint mutation between reference residues
<400>73
aatatggaag gggattccaa atctgaaggg acctcagggg 40
<210>74
<211>3375
<212>DNA
<213> Artificial sequence
<220>
<223> wild type of JAK3 cDNA
<400>74
atggcacctc caagtgaaga gacgcccctg atccctcagc gttcatgcag cctcttgtcc 60
acggaggctg gtgccctgca tgtgctgctg cccgctcggg gccccgggcc cccccagcgc 120
ctatctttct cctttgggga ccacttggct gaggacctgt gcgtgcaggc tgccaaggcc 180
agcggcatcc tgcctgtgta ccactccctc tttgctctgg ccacggagga cctgtcctgc 240
tggttccccc cgagccacat cttctccgtg gaggatgcca gcacccaagt cctgctgtac 300
aggattcgct tttacttccc caattggttt gggctggaga agtgccaccg cttcgggcta 360
cgcaaggatt tggccagtgc tatccttgac ctgccagtcc tggagcacct ctttgcccag 420
caccgcagtg acctggtgag tgggcgcctc cccgtgggcc tcagtctcaa ggagcagggt 480
gagtgtctca gcctggccgt gttggacctg gcccggatgg cgcgagagca ggcccagcgg 540
ccgggagagc tgctgaagac tgtcagctac aaggcctgcc tacccccaag cctgcgcgac 600
ctgatccagg gcctgagctt cgtgacgcgg aggcgtattc ggaggacggt gcgcagagcc 660
ctgcgccgcg tggccgcctg ccaggcagac cggcactcgc tcatggccaa gtacatcatg 720
gacctggagc ggctggatcc agccggggcc gccgagacct tccacgtggg cctccctggg 780
gcccttggtg gccacgacgg gctggggctg ctccgcgtgg ctggtgacgg cggcatcgcc 840
tggacccagg gagaacagga ggtcctccag cccttctgcg actttccaga aatcgtagac 900
attagcatca agcaggcccc gcgcgttggc ccggccggag agcaccgcct ggtcactgtt 960
accaggacag acaaccagat tttagaggcc gagttcccag ggctgcccga ggctctgtcg 1020
ttcgtggcgc tcgtggacgg ctacttccgg ctgaccacgg actcccagca cttcttctgc 1080
aaggaggtgg caccgccgag gctgctggag gaagtggccg agcagtgcca cggccccatc 1140
actctggact ttgccatcaa caagctcaag actgggggct cacgtcctgg ctcctatgtt 1200
ctccgccgca gcccccagga ctttgacagc ttcctcctca ctgtctgtgt ccagaacccc 1260
cttggtcctg attataaggg ctgcctcatc cggcgcagcc ccacaggaac cttccttctg 1320
gttggcctca gccgacccca cagcagtctt cgagagctcc tggcaacctg ctgggatggg 1380
gggctgcacg tagatggggt ggcagtgacc ctcacttcct gctgtatccc cagacccaaa 1440
gaaaagtcca acctgatcgt ggtccagaga ggtcacagcc cacccacatc atccttggtt 1500
cagccccaat cccaatacca gctgagtcag atgacatttc acaagatccc tgctgacagc 1560
ctggagtggc atgagaacct gggccatggg tccttcacca agatttaccg gggctgtcgc 1620
catgaggtgg tggatgggga ggcccgaaag acagaggtgc tgctgaaggt catggatgcc 1680
aagcacaaga actgcatgga gtcattcctg gaagcagcga gcttgatgag ccaagtgtcg 1740
taccggcatc tcgtgctgct ccacggcgtg tgcatggctg gagacagcac catggtgcag 1800
gaatttgtac acctgggggc catagacatg tatctgcgaa aacgtggcca cctggtgcca 1860
gccagctgga agctgcaggt ggtcaaacag ctggcctacg ccctcaacta tctggaggac 1920
aaaggcctgc cccatggcaa tgtctctgcc cggaaggtgc tcctggctcg ggagggggct 1980
gatgggagcc cgcccttcat caagctgagt gaccctgggg tcagccccgc tgtgttaagc 2040
ctggagatgc tcaccgacag gatcccctgg gtggcccccg agtgtctccg ggaggcgcag 2100
acacttagct tggaagctga caagtggggc ttcggcgcca cggtctggga agtgtttagt 2160
ggcgtcacca tgcccatcag tgccctggat cctgctaaga aactccaatt ttatgaggac 2220
cggcagcagc tgccggcccc caagtggaca gagctggccc tgctgattca acagtgcatg 2280
gcctatgagc cggtccagag gccctccttc cgagccgtca ttcgtgacct caatagcctc 2340
atctcttcag actatgagct cctctcagac cccacacctg gtgccctggc acctcgtgat 2400
gggctgtgga atggtgccca gctctatgcc tgccaagacc ccacgatctt cgaggagaga 2460
cacctcaagt acatctcaca gctgggcaag ggcaactttg gcagcgtgga gctgtgccgc 2520
tatgacccgc taggcgacaa tacaggtgcc ctggtggccg tgaaacagct gcagcacagc 2580
gggccagacc agcagaggga ctttcagcgg gagattcaga tcctcaaagc actgcacagt 2640
gatttcattg tcaagtatcg tggtgtcagc tatggcccgg gccgccagag cctgcggctg 2700
gtcatggagt acctgcccag cggctgcttg cgcgacttcc tgcagcggca ccgcgcgcgc 2760
ctcgatgcca gccgcctcct tctctattcc tcgcagatct gcaagggcat ggagtacctg 2820
ggctcccgcc gctgcgtgca ccgcgacctg gccgcccgaa acatcctcgt ggagagcgag 2880
gcacacgtca agatcgctga cttcggccta gctaagctgc tgccgcttga caaagactac 2940
tacgtggtcc gcgagccagg ccagagcccc attttctggt atgcccccga atccctctcg 3000
gacaacatct tctctcgcca gtcagacgtc tggagcttcg gggtcgtcct gtacgagctc 3060
ttcacctact gcgacaaaag ctgcagcccc tcggccgagt tcctgcggat gatgggatgt 3120
gagcgggatg tccccgccct ctgccgcctc ttggaactgc tggaggaggg ccagaggctg 3180
ccggcgcctc ctgcctgccc tgctgaggtt cacgagctca tgaagctgtg ctgggcccct 3240
agcccacagg accggccatc attcagcgcc ctgggccccc agctggacat gctgtggagc 3300
ggaagccggg ggtgtgagac tcatgccttc actgctcacc cagagggcaa acaccactcc 3360
ctgtccttttcatag 3375
<210>75
<211>3375
<212>DNA
<213> Artificial sequence
<220>
<223> JAK3 cDNA Single mutation 1
<400>75
atggcacctc caagtgaaga gacgcccctg atccctcagc gttcatgcag cctcttgtcc 60
acggaggctg gtgccctgca tgtgctgctg cccgctcggg gccccgggcc cccccagcgc 120
ctatctttct cctttgggga ccacttggct gaggacctgt gcgtgcaggc tgccaaggcc 180
agcggcatcc tgcctgtgta ccactccctc tttgctctgg ccacggagga cctgtcctgc 240
tggttccccc cgagccacat cttctccgtg gaggatgcca gcacccaagt cctgctgtac 300
aggattcgct tttacttccc caattggttt gggctggaga agtgccaccg cttcgggcta 360
cgcaaggatt tggccagtgc tatccttgac ctgccagtcc tggagcacct ctttgcccag 420
caccgcagtg acctggtgag tgggcgcctc cccgtgggcc tcagtctcaa ggagcagggt 480
gagtgtctca gcctggccgt gttggacctg gcccggatgg cgcgagagca ggcccagcgg 540
ccgggagagc tgctgaagac tgtcagctac aaggcctgcc tacccccaag cctgcgcgac 600
ctgatccagg gcctgagctt cgtgacgcgg aggcgtattc ggaggacggt gcgcagagcc 660
ctgcgccgcg tggccgcctg ccaggcagac cggcactcgc tcatggccaa gtacatcatg 720
gacctggagc ggctggatcc agccggggcc gccgagacct tccacgtggg cctccctggg 780
gcccttggtg gccacgacgg gctggggctg ctccgcgtgg ctggtgacgg cggcatcgcc 840
tggacccagg gagaacagga ggtcctccag cccttctgcg actttccaga aatcgtagac 900
attagcatca agcaggcccc gcgcgttggc ccggccggag agcaccgcct ggtcactgtt 960
accaggacag acaaccagat tttagaggcc gagttcccag ggctgcccga ggctctgtcg 1020
ttcgtggcgc tcgtggacgg ctacttccgg ctgaccacgg actcccagca cttcttctgc 1080
aaggaggtgg caccgccgag gctgctggag gaagtggccg agcagtgcca cggccccatc 1140
actctggact ttgccatcaa caagctcaag actgggggct cacgtcctgg ctcctatgtt 1200
ctccgccgca gcccccagga ctttgacagc ttcctcctca ctgtctgtgt ccagaacccc 1260
cttggtcctg attataaggg ctgcctcatc cggcgcagcc ccacaggaac cttccttctg 1320
gttggcctca gccgacccca cagcagtctt cgagagctcc tggcaacctg ctgggatggg 1380
gggctgcacg tagatggggt ggcagtgacc ctcacttcct gctgtatccc cagacccaaa 1440
gaaaagtcca acctgatcgt ggtccagaga ggtcacagcc cacccacatc atccttggtt 1500
cagccccaat cccaatacca gctgagtcag atgacatttc acaagatccc tgctgacagc 1560
ctggagtggc atgagaacct gggccatggg tccttcacca agatttaccg gggctgtcgc 1620
catgaggtgg tggatgggga ggcccgaaag acagaggtgc tgctgaaggt catggatgcc 1680
aagcacaaga actgcatgga gtcattcctg gaagtagcga gcttgatgag ccaagtgtcg 1740
taccggcatc tcgtgctgct ccacggcgtg tgcatggctg gagacagcac catggtgcag 1800
gaatttgtac acctgggggc catagacatg tatctgcgaa aacgtggcca cctggtgcca 1860
gccagctgga agctgcaggt ggtcaaacag ctggcctacg ccctcaacta tctggaggac 1920
aaaggcctgc cccatggcaa tgtctctgcc cggaaggtgc tcctggctcg ggagggggct 1980
gatgggagcc cgcccttcat caagctgagt gaccctgggg tcagccccgc tgtgttaagc 2040
ctggagatgc tcaccgacag gatcccctgg gtggcccccg agtgtctccg ggaggcgcag 2100
acacttagct tggaagctga caagtggggc ttcggcgcca cggtctggga agtgtttagt 2160
ggcgtcacca tgcccatcag tgccctggat cctgctaaga aactccaatt ttatgaggac 2220
cggcagcagc tgccggcccc caagtggaca gagctggccc tgctgattca acagtgcatg 2280
gcctatgagc cggtccagag gccctccttc cgagccgtca ttcgtgacct caatagcctc 2340
atctcttcag actatgagct cctctcagac cccacacctg gtgccctggc acctcgtgat 2400
gggctgtgga atggtgccca gctctatgcc tgccaagacc ccacgatctt cgaggagaga 2460
cacctcaagt acatctcaca gctgggcaag ggcaactttg gcagcgtgga gctgtgccgc 2520
tatgacccgc taggcgacaa tacaggtgcc ctggtggccg tgaaacagct gcagcacagc 2580
gggccagacc agcagaggga ctttcagcgg gagattcaga tcctcaaagc actgcacagt 2640
gatttcattg tcaagtatcg tggtgtcagc tatggcccgg gccgccagag cctgcggctg 2700
gtcatggagt acctgcccag cggctgcttg cgcgacttcc tgcagcggca ccgcgcgcgc 2760
ctcgatgcca gccgcctcct tctctattcc tcgcagatct gcaagggcat ggagtacctg 2820
ggctcccgcc gctgcgtgca ccgcgacctg gccgcccgaa acatcctcgt ggagagcgag 2880
gcacacgtca agatcgctga cttcggccta gctaagctgc tgccgcttga caaagactac 2940
tacgtggtcc gcgagccagg ccagagcccc attttctggt atgcccccga atccctctcg 3000
gacaacatct tctctcgcca gtcagacgtc tggagcttcg gggtcgtcct gtacgagctc 3060
ttcacctact gcgacaaaag ctgcagcccc tcggccgagt tcctgcggat gatgggatgt 3120
gagcgggatg tccccgccct ctgccgcctc ttggaactgc tggaggaggg ccagaggctg 3180
ccggcgcctc ctgcctgccc tgctgaggtt cacgagctca tgaagctgtg ctgggcccct 3240
agcccacagg accggccatc attcagcgcc ctgggccccc agctggacat gctgtggagc 3300
ggaagccggg ggtgtgagac tcatgccttc actgctcacc cagagggcaa acaccactcc 3360
ctgtcctttt catag 3375
<210>76
<211>3375
<212>DNA
<213> Artificial sequence
<220>
<223> JAK3 cDNA Single mutation 2
<400>76
atggcacctc caagtgaaga gacgcccctg atccctcagc gttcatgcag cctcttgtcc 60
acggaggctg gtgccctgca tgtgctgctg cccgctcggg gccccgggcc cccccagcgc 120
ctatctttct cctttgggga ccacttggct gaggacctgt gcgtgcaggc tgccaaggcc 180
agcggcatcc tgcctgtgta ccactccctc tttgctctgg ccacggagga cctgtcctgc 240
tggttccccc cgagccacat cttctccgtg gaggatgcca gcacccaagt cctgctgtac 300
aggattcgct tttacttccc caattggttt gggctggaga agtgccaccg cttcgggcta 360
cgcaaggatt tggccagtgc tatccttgac ctgccagtcc tggagcacct ctttgcccag 420
caccgcagtg acctggtgag tgggcgcctc cccgtgggcc tcagtctcaa ggagcagggt 480
gagtgtctca gcctggccgt gttggacctg gcccggatgg cgcgagagca ggcccagcgg 540
ccgggagagc tgctgaagac tgtcagctac aaggcctgcc tacccccaag cctgcgcgac 600
ctgatccagg gcctgagctt cgtgacgcgg aggcgtattc ggaggacggt gcgcagagcc 660
ctgcgccgcg tggccgcctg ccaggcagac cggcactcgc tcatggccaa gtacatcatg 720
gacctggagc ggctggatcc agccggggcc gccgagacct tccacgtggg cctccctggg 780
gcccttggtg gccacgacgg gctggggctg ctccgcgtgg ctggtgacgg cggcatcgcc 840
tggacccagg gagaacagga ggtcctccag cccttctgcg actttccaga aatcgtagac 900
attagcatca agcaggcccc gcgcgttggc ccggccggag agcaccgcct ggtcactgtt 960
accaggacag acaaccagat tttagaggcc gagttcccag ggctgcccga ggctctgtcg 1020
ttcgtggcgc tcgtggacgg ctacttccgg ctgaccacgg actcccagca cttcttctgc 1080
aaggaggtgg caccgccgag gctgctggag gaagtggccg agcagtgcca cggccccatc 1140
actctggact ttgccatcaa caagctcaag actgggggct cacgtcctgg ctcctatgtt 1200
ctccgccgca gcccccagga ctttgacagc ttcctcctca ctgtctgtgt ccagaacccc 1260
cttggtcctg attataaggg ctgcctcatc cggcgcagcc ccacaggaac cttccttctg 1320
gttggcctca gccgacccca cagcagtctt cgagagctcc tggcaacctg ctgggatggg 1380
gggctgcacg tagatggggt ggcagtgacc ctcacttcct gctgtatccc cagacccaaa 1440
gaaaagtcca acctgatcgt ggtccagaga ggtcacagcc cacccacatc atccttggtt 1500
cagccccaat cccaatacca gctgagtcag atgacatttc acaagatccc tgctgacagc 1560
ctggagtggc atgagaacct gggccatggg tccttcacca agatttaccg gggctgtcgc 1620
catgaggtgg tggatgggga ggcccgaaag acagaggtgc tgctgaaggt catggatgcc 1680
aagcacaaga actgcatgga gtcattcctg gaagcagtga gcttgatgag ccaagtgtcg 1740
taccggcatc tcgtgctgct ccacggcgtg tgcatggctg gagacagcac catggtgcag 1800
gaatttgtac acctgggggc catagacatg tatctgcgaa aacgtggcca cctggtgcca 1860
gccagctgga agctgcaggt ggtcaaacag ctggcctacg ccctcaacta tctggaggac 1920
aaaggcctgc cccatggcaa tgtctctgcc cggaaggtgc tcctggctcg ggagggggct 1980
gatgggagcc cgcccttcat caagctgagt gaccctgggg tcagccccgc tgtgttaagc 2040
ctggagatgc tcaccgacag gatcccctgg gtggcccccg agtgtctccg ggaggcgcag 2100
acacttagct tggaagctga caagtggggc ttcggcgcca cggtctggga agtgtttagt 2160
ggcgtcacca tgcccatcag tgccctggat cctgctaaga aactccaatt ttatgaggac 2220
cggcagcagc tgccggcccc caagtggaca gagctggccc tgctgattca acagtgcatg 2280
gcctatgagc cggtccagag gccctccttc cgagccgtca ttcgtgacct caatagcctc 2340
atctcttcag actatgagct cctctcagac cccacacctg gtgccctggc acctcgtgat 2400
gggctgtgga atggtgccca gctctatgcc tgccaagacc ccacgatctt cgaggagaga 2460
cacctcaagt acatctcaca gctgggcaag ggcaactttg gcagcgtgga gctgtgccgc 2520
tatgacccgc taggcgacaa tacaggtgcc ctggtggccg tgaaacagct gcagcacagc 2580
gggccagacc agcagaggga ctttcagcgg gagattcaga tcctcaaagc actgcacagt 2640
gatttcattg tcaagtatcg tggtgtcagc tatggcccgg gccgccagag cctgcggctg 2700
gtcatggagt acctgcccag cggctgcttg cgcgacttcc tgcagcggca ccgcgcgcgc 2760
ctcgatgcca gccgcctcct tctctattcc tcgcagatct gcaagggcat ggagtacctg 2820
ggctcccgcc gctgcgtgca ccgcgacctg gccgcccgaa acatcctcgt ggagagcgag 2880
gcacacgtca agatcgctga cttcggccta gctaagctgc tgccgcttga caaagactac 2940
tacgtggtcc gcgagccagg ccagagcccc attttctggt atgcccccga atccctctcg 3000
gacaacatct tctctcgcca gtcagacgtc tggagcttcg gggtcgtcct gtacgagctc 3060
ttcacctact gcgacaaaag ctgcagcccc tcggccgagt tcctgcggat gatgggatgt 3120
gagcgggatg tccccgccct ctgccgcctc ttggaactgc tggaggaggg ccagaggctg 3180
ccggcgcctc ctgcctgccc tgctgaggtt cacgagctca tgaagctgtg ctgggcccct 3240
agcccacagg accggccatc attcagcgcc ctgggccccc agctggacat gctgtggagc 3300
ggaagccggg ggtgtgagac tcatgccttc actgctcacc cagagggcaa acaccactcc 3360
ctgtcctttt catag 3375
<210>77
<211>3375
<212>DNA
<213> Artificial sequence
<220>
<223> JAK3 cDNA double mutation
<400>77
atggcacctc caagtgaaga gacgcccctg atccctcagc gttcatgcag cctcttgtcc 60
acggaggctg gtgccctgca tgtgctgctg cccgctcggg gccccgggcc cccccagcgc 120
ctatctttct cctttgggga ccacttggct gaggacctgt gcgtgcaggc tgccaaggcc 180
agcggcatcc tgcctgtgta ccactccctc tttgctctgg ccacggagga cctgtcctgc 240
tggttccccc cgagccacat cttctccgtg gaggatgcca gcacccaagt cctgctgtac 300
aggattcgct tttacttccc caattggttt gggctggaga agtgccaccg cttcgggcta 360
cgcaaggatt tggccagtgc tatccttgac ctgccagtcc tggagcacct ctttgcccag 420
caccgcagtg acctggtgag tgggcgcctc cccgtgggcc tcagtctcaa ggagcagggt 480
gagtgtctca gcctggccgt gttggacctg gcccggatgg cgcgagagca ggcccagcgg 540
ccgggagagc tgctgaagac tgtcagctac aaggcctgcc tacccccaag cctgcgcgac 600
ctgatccagg gcctgagctt cgtgacgcgg aggcgtattc ggaggacggt gcgcagagcc 660
ctgcgccgcg tggccgcctg ccaggcagac cggcactcgc tcatggccaa gtacatcatg 720
gacctggagc ggctggatcc agccggggcc gccgagacct tccacgtggg cctccctggg 780
gcccttggtg gccacgacgg gctggggctg ctccgcgtgg ctggtgacgg cggcatcgcc 840
tggacccagg gagaacagga ggtcctccag cccttctgcg actttccaga aatcgtagac 900
attagcatca agcaggcccc gcgcgttggc ccggccggag agcaccgcct ggtcactgtt 960
accaggacag acaaccagat tttagaggcc gagttcccag ggctgcccga ggctctgtcg 1020
ttcgtggcgc tcgtggacgg ctacttccgg ctgaccacgg actcccagca cttcttctgc 1080
aaggaggtgg caccgccgag gctgctggag gaagtggccg agcagtgcca cggccccatc 1140
actctggact ttgccatcaa caagctcaag actgggggct cacgtcctgg ctcctatgtt 1200
ctccgccgca gcccccagga ctttgacagc ttcctcctca ctgtctgtgt ccagaacccc 1260
cttggtcctg attataaggg ctgcctcatc cggcgcagcc ccacaggaac cttccttctg 1320
gttggcctca gccgacccca cagcagtctt cgagagctcc tggcaacctg ctgggatggg 1380
gggctgcacg tagatggggt ggcagtgacc ctcacttcct gctgtatccc cagacccaaa 1440
gaaaagtcca acctgatcgt ggtccagaga ggtcacagcc cacccacatc atccttggtt 1500
cagccccaat cccaatacca gctgagtcag atgacatttc acaagatccc tgctgacagc 1560
ctggagtggc atgagaacct gggccatggg tccttcacca agatttaccg gggctgtcgc 1620
catgaggtgg tggatgggga ggcccgaaag acagaggtgc tgctgaaggt catggatgcc 1680
aagcacaaga actgcatgga gtcattcctg gaagtagtga gcttgatgag ccaagtgtcg 1740
taccggcatc tcgtgctgct ccacggcgtg tgcatggctg gagacagcac catggtgcag 1800
gaatttgtac acctgggggc catagacatg tatctgcgaa aacgtggcca cctggtgcca 1860
gccagctgga agctgcaggt ggtcaaacag ctggcctacg ccctcaacta tctggaggac 1920
aaaggcctgc cccatggcaa tgtctctgcc cggaaggtgc tcctggctcg ggagggggct 1980
gatgggagcc cgcccttcat caagctgagt gaccctgggg tcagccccgc tgtgttaagc 2040
ctggagatgc tcaccgacag gatcccctgg gtggcccccg agtgtctccg ggaggcgcag 2100
acacttagct tggaagctga caagtggggc ttcggcgcca cggtctggga agtgtttagt 2160
ggcgtcacca tgcccatcag tgccctggat cctgctaaga aactccaatt ttatgaggac 2220
cggcagcagc tgccggcccc caagtggaca gagctggccc tgctgattca acagtgcatg 2280
gcctatgagc cggtccagag gccctccttc cgagccgtca ttcgtgacct caatagcctc 2340
atctcttcag actatgagct cctctcagac cccacacctg gtgccctggc acctcgtgat 2400
gggctgtgga atggtgccca gctctatgcc tgccaagacc ccacgatctt cgaggagaga 2460
cacctcaagt acatctcaca gctgggcaag ggcaactttg gcagcgtgga gctgtgccgc 2520
tatgacccgc taggcgacaa tacaggtgcc ctggtggccg tgaaacagct gcagcacagc 2580
gggccagacc agcagaggga ctttcagcgg gagattcaga tcctcaaagc actgcacagt 2640
gatttcattg tcaagtatcg tggtgtcagc tatggcccgg gccgccagag cctgcggctg 2700
gtcatggagt acctgcccag cggctgcttg cgcgacttcc tgcagcggca ccgcgcgcgc 2760
ctcgatgcca gccgcctcct tctctattcc tcgcagatct gcaagggcat ggagtacctg 2820
ggctcccgcc gctgcgtgca ccgcgacctg gccgcccgaa acatcctcgt ggagagcgag 2880
gcacacgtca agatcgctga cttcggccta gctaagctgc tgccgcttga caaagactac 2940
tacgtggtcc gcgagccagg ccagagcccc attttctggt atgcccccga atccctctcg 3000
gacaacatct tctctcgcca gtcagacgtc tggagcttcg gggtcgtcct gtacgagctc 3060
ttcacctact gcgacaaaag ctgcagcccc tcggccgagt tcctgcggat gatgggatgt 3120
gagcgggatg tccccgccct ctgccgcctc ttggaactgc tggaggaggg ccagaggctg 3180
ccggcgcctc ctgcctgccc tgctgaggtt cacgagctca tgaagctgtg ctgggcccct 3240
agcccacagg accggccatc attcagcgcc ctgggccccc agctggacat gctgtggagc 3300
ggaagccggg ggtgtgagac tcatgccttc actgctcacc cagagggcaa acaccactcc 3360
ctgtcctttt catag 3375
<210>78
<211>20065
<212>DNA
<213> Artificial sequence
<220>
<223> PD-L1 gDNA full length, > hg19_ dna range = chr9:5450503-5470567
5'pad = 03' pad =0 chain = + repeated Masking = none
<400>78
ggcgcaacgc tgagcagctg gcgcgtcccg cgcggcccca gttctgcgca gcttcccgag 60
gctccgcacc agccgcgctt ctgtccgcct gcaggtaggg agcgttgttc ctccgcgggt 120
gcccacggcc cagtatctct ggctagctcg ctgggcactt taggacggag ggtctctaca 180
ccctttcttt gggatggaga gaggagaagg gaaagggaac gcgatggtct agggggcagt 240
agagccaatt acctgttggg gttaataaga acaggcaatg catctggcct tcctccaggc 300
gcgattcagt tttgctctaa aaataattta tacctctaaa aataaataag ataggtagta 360
taggataggt agtcattctt atgcgactgt gtgttcagaa tatagctctg atgctaggct 420
ggaggtctgg acacgggtcc aagtccaccg ccagctgctt gctagtaaca tgacttgtgt 480
aagttatccc agctgcagca tctaagtaag tctcttcctg cgctaagcag gtccaggatc 540
cctgaacgga atttatttgc tctgtccatt ctgagaaccc aaaggagtcc taaaagagga 600
atggaggagc ctaagaataa aaatagtata ataaaacatt tcttagacac attgaccttg 660
gcctatgtca aagttcagtc tgggtttgtc ttataacaca aggagtaaaa gtaccattgt 720
tctacctctt tttttaatac ttgaaaaaaa tttactgtgg atgcttttct atgaattaaa 780
taaccttcta aaaaatgttt tcattgctgc attcgattag attgggtaac taaatgaaat 840
taattcctca ctgttgggta taaaggttat ttacagtggt tctgtcttag ccattcactg 900
aactcattgc atatatatct ctggaatatt gctgattgtt tccttcaagt aaacttagaa 960
gtgtaactac ttagtcaaag agcctgaata ttttaaaggc cttttgaaga aaactgaaaa 1020
tgctttccag aaaggatgta tcagttgaca atgacagtcg tcaacagtat ttaaggagaa 1080
ctatgatact ctgaagaaaa acttagcctt tctcagtaaa agtaggtagg cagaggccac 1140
atgacagcag ttagagtgtg gtcttcaagg aagtcacaga aatactgtgg ggaattgaaa 1200
ccccatgtgg aaaatgtaca agagtgtctc agtgtgactg agaaggaggt tgggcatggg 1260
gtttcatgga gtttaataaa gtttggtcac ttagtagagg tttaataaat caactgtctt 1320
aatctttgat cctacttaag aatttttttt ttgtttttgt agagatgggg ctcttgttat 1380
gttgcccagg ctgttctcga actcctagcc tcaggcgatc ctccctcctc aggctccaga 1440
agtcctggga ttactggcgg gagccaccat gcaggcctct tgctcctact tttgagaaag 1500
gaagtttaac cggttttttt tgtctttttt tttttttttt tgagacagag tctcactctg 1560
ttgcccatgc tggagtgcag tggtgcaatc tcagctcact gcctcccggg ttcaagtgat 1620
tctcctgcct cagcctcccg agtagctggg actacaggca cctgccacca cgcccagcta 1680
atttttgtat ttttagtaga aatggggttt caccatattg gccaggctga tctcgaactc 1740
ctgacctcag gtgatccgcc tgcctcggcc tcccaaagtg ctgggattac aggcatgagc 1800
cactgctcct ggctgcttaa ctttttctct atctcatcct cctacccatc ctacccttgg 1860
aagatagaga agtagtatta gttccatagt gttatactgg gcttccccca gggacaaacc 1920
cacttcccca acctgaatga gccatcactt cttccccagt ttacatttca ttgctcttta 1980
aatgtctcca ttcggatatg ggaattcaca tatggtcata attcttacct gaagaagatg 2040
tcagtcttct tctcttagac caactgccct gatatgaggt ttagaggtta aagaacatgt 2100
gtgtatttac atgatctttg tattctgcct tttcgtccct cactaatgac agctgcaccc 2160
caaggaaatg gagctgtgga agagagggtt tgataagaaa ttaagtaaat attggatcta 2220
atccatcacc ctccaggaag cctttattac tcctaaaaat ttcaaccaaa ttcattaaag 2280
gacaagaact ccaccagagt aggccataaa cattggcaaa attagttgta atccatgact 2340
agatttaatg tccctttgtt ttattcccat atggttataa tgctttgctt ggcattaggg 2400
gtattttaag ttttcttctg cctagtaagt gaatttgtgt ttataataca ataatcataa 2460
aatatcacat taatatttta taactgtaca gttataaaat attttataag taatatttat 2520
attttataag taatatttta taactgtaca gttaactctg gcccaaggaa aagatagtct 2580
gatagatgct gcagccccat tttagcaaat gtgacctcac aggcctgaat gccatcgcta 2640
ttccacatct acaggataga cggaaaggaa agaaataaaa aaataggtac ctaacactgg 2700
caagaggatg atgactcatg ttatttcact taaccttttt atcttttaac atgaaggact 2760
catacaggtt gataagaaac cagtgacata aacagaccaa aaaatgatca gatctttcaa 2820
attagcaaaa aaataatatt ttttaaacaa tgggtgaaaa tacagtgtaa cagtaccaat 2880
tatcaacatg tgttgagaac cagaaaaatg ttctttttct ttgatcagca acactatttg 2940
ggaaaatcta tcctcagggc ctagcctggg gccctggcac acagtaggca ctcaacgaat 3000
atttgctgaa cacacaaata cttatgatat tttaaaaaat tggcaacaat ctgataccta 3060
acaatagagg gattaaatat tatggaactg ttaaataaga tgcttatgaa taccatgcag 3120
taagatgggc aatatttatg ccataagctt taatgaaaca aatgggtatt aaatgtatga 3180
taaggttata aattactttt taaaagatta cagggaaaaa aattgaaaga tatacactga 3240
aatgtttttt gctcacagtg gtgacaaggt ttctcagcac tggcactgtt gacgttttag 3300
gctgtatgtc tttgctgtgg gaggctggcc tgtgcactgc agggtgtttg gcagcactct 3360
tggcctctgc ccctagatag caatagcagt cctccctcaa ccagcccaat tttgacaacc 3420
aaaaatgttt ccaggcatca ccagatgctc cctgggtgag agtgatgaaa tagtagggga 3480
ttttcccctt cttttcttat tttctgtaat tccattatat tactttaata ataaagaaaa 3540
aaacataaaa aataaacgaa tgttattatt ctacgtcagt ttggatgttt ggactccatt 3600
ttggggttct ttccattata tcacttggtc tgctaaacat tctacggttt ggtaaggtga 3660
agtgattcat gaaattttgg ttttattttt ttcctgatac taaaaataaa acattctttc 3720
acttggaaat ttggacacag aacaccaaaa aaaatccata atctcatctc tctttttctg 3780
tcttttcctt ccttttttcc ctttaaaaac aataaagagt gaaacctacc tgttctccct 3840
ctaatttaat tcctaaatat aatcactgtc aatatcttgg acatttcctg tgtctaaaca 3900
cacacacaca cttttttttt tcagcaaaag tggatttctg ctacatgtag tgttctgcaa 3960
cttactttct atgtgtttac aaaatcagta catgtacata tgctgaattc agtccttaat 4020
ggtattatat tttgtgaata taccaaaatt tgtttaacca cttagacaat ctaggatatt 4080
ctcagtttgc tgttatgagc aatgctcttc ctttacatat acagacatat atatatatat 4140
gtgtgtgtgt gtgtttttgt tttagtagga tagatttcta ggagagggtg aaaggtctta 4200
tgacatccgc atttacgatt gtaataggaa gtatcaaagt gccccctaaa gaaaaaaatc 4260
ctcccattag tgggtaagaa agcctatttg ttcatatctt cacaaacact aaatattaga 4320
aatatttaca attgtggtca agctcataag tgaaaatggt atttcatatc ttatattttt 4380
tattgtgaga ttgaacatct ttcatatgtt tacatgtcac ctgtatttct tattctctga 4440
actatatgtt atgacctttc actttttttc ctcatgggtt atgtgtagtt tgtatagttg 4500
tcttattgat tgttaggagc tatttatata ttaggaacat taatctcctg tcttatatat 4560
acgtggcatc gattagttga tcatttgtga gttcatgtct gtatacaaag attggagagg 4620
cactaagagg gaaaacttac ctctttctta tcaaagtttg taaatatatg tataacagaa 4680
gagggagaaa atattaataa atgcacagat tggctgaaat agagtataaa tcttttactc 4740
ccctacttca acataaactg caaaaggaga gtgacttttc tttcactctg acttccgtat 4800
tcctcatgct taaaatagtg cctagcacag aagaggtgct caatcagtgt ttgctaaacg 4860
aaataattag tcacatttca agcaggatga ctaaatgaag aatagaatct aggcagatac 4920
tctggaagag tggctgtgag tcattcatat cttagtatga attagtcaaa tccaactctc 4980
tccccttccc actccccact gttagtagaa gaatctgttt attgagagaa tagatttata 5040
atttagaata agtgagaggg gcagaagagg agattttgaa ggatggcacc tgaaggagga 5100
ctagcatggc tgagacagtg aagtggaagc cttgaatagc taaagggtaa gatgaaagta 5160
tttagctgta gggggaaaaa gcattgacag gttggaaaag taaaagtcag attctccttg 5220
ctctgaaatt ttgtacaggg caggttctac taggtatgtt acaatgcaga aaaaacatga 5280
aataattgag aggaatttgg tgcaatatta tcttcttggc ttcttttgag tgggcagatt 5340
tttttcacgg cctgtaacta taataaattt gaaacttctc atcttttagt aacttttttc 5400
acttaagttt atgtggctgt gggcaatgga atgaagatat tgaacttcca attccctgtt 5460
gggtttccac aattacaagt caatcatgac tggttattag aagactattt cagttagaac 5520
caccaagtcc catattgtca tattgtatgt ttaattatta agtgaagcag tcttcttttc 5580
gtgttttcca taattagggc attccagaaa gatgaggata tttgctgtct ttatattcat 5640
gacctactgg catttgctga acggtaagac accaaatcct tccattaggt tctatatttt 5700
aaatatttta accatgagtt taaaactaaa atgatcattt aaaatgcatg caattttctt 5760
atagagagaa cattctattc tttcttctac tttacacaat ggcaaagtct tctttctact 5820
ttacgcaatg ataaagttac ctgtgtcatt ttgtaaaaat atagagaata tagacaaatt 5880
gaaagacaca aaataatcta ttacccattt cccagggtta actactgaaa atatctgggg 5940
aaatggcctg tatgtataca tttatttgtt tgctttcaac aaggccaaga tcctttgatc 6000
tttcagtctt ggttgctctg tgacatgcct ttcctgatga ggatacttta aggaagaatt 6060
gtaagataca tggaaaatgt caggctaaca cagtactggc atcaccctgt gctctttcct 6120
gaactccata ccaatgtact tcttgccaga aaactgatca aaagtttagg gaagtaaaaa 6180
gagatgactg ttagaatcta ccattccctc tatgtaggaa gcaaataggt gtcctgtcaa 6240
aggacattct ggggatgtct acatgaaacc aactctccct ggttgtaagg actccatctc 6300
catataatat ttatacagta atatatgttt ataaattgtg ggggcaactt gtttagctaa 6360
ttttattatt ctgctattgg gacactgtgt ctcagcatga gatatagtgt cccaaaacat 6420
atttcaagcc cattggataa aatatgtgtt tagcaagttc ttaaatataa tgataacata 6480
accgaccaga taaagtgatt tataaacgct gtgccaattt tgtaaatgtt tcgaggaatt 6540
ttcccttttc tgaagattgt ccttctttct ttttagcatt tactgtcacg gttcccaagg 6600
acctatatgt ggtagagtat ggtagcaata tgacaattga atgcaaattc ccagtagaaa 6660
aacaattaga cctggctgca ctaattgtct attgggaaat ggaggataag aacattattc 6720
aatttgtgca tggagaggaa gacctgaagg ttcagcatag tagctacaga cagagggccc 6780
ggctgttgaa ggaccagctc tccctgggaa atgctgcact tcagatcaca gatgtgaaat 6840
tgcaggatgc aggggtgtac cgctgcatga tcagctatgg tggtgccgac tacaagcgaa 6900
ttactgtgaa agtcaatggt aagaattatt atagatgaga ggcctgatct ttattgaaaa 6960
catattccaa gtgttgaaga cttttcattc ttgtaagtcc atacttattt tcaaacagaa 7020
cagcatagtc tgttcattca ttcattcaat tcatgaattc attcacataa ttatccaatt 7080
tcttgagcac ctatttgata gtcactggaa atccagagac aaacaacaca gagccatgtt 7140
ctacagtatg tacagttttc caaaaagaat ttctagtctt tactttttta ttacaaatgg 7200
aatacgtata cttgcaaata attcagatac tgtggaagag atcaaatgaa ttgcaaaagt 7260
gtccctcctc ccttcaccac tatctcccat ggcatgcaga gagagtaacc attatttgtg 7320
tgtccctcca gaaatttttt tattcaacta ctattttttt attttattag gtccgtcagt 7380
tttccttttt tgagcctctc tatatcaaat gcaaataaat atattcagaa caaaccccac 7440
tgtaaggttc acattaaaaa agacttgaag tcaccctatg aagacaaaaa ataatcacat 7500
taagtgtgaa agaacctatt cttccagtac aggataagcc atacttactg ggcatatatt 7560
catcttgaaa atctatactg atgttgtctt ggggaattga aaaggaacta ggagtgttag 7620
ttcctcggta ttgacccaca gttatgttat caggtcactt gagttcaaag ttttgtgttg 7680
gcactagcta agtaaaggaa aacacctctg ctttcattgt tgagtttcac agaattgaga 7740
gctgaaagga tcccaggcag gagcagctaa tccaaactcc cacaaagaac aaaaatcccc 7800
cagaggatct tctgttctta tatttcctgc aatggcgtcc ctgtcatatc ccacaatggc 7860
ctccctgcca tttggatatc ccttccatat cctgttgaaa ttactcccta atagtaagct 7920
gaaatctgcc cctctagttg tagtcttggg attatttcat ttacatgatg accttttaat 7980
atttgactag aattaaatca tctccccttg gtctttccat tcctgggcta actaccatca 8040
atctgagggc taacaataca agtagaaaaa gtatacattt gtcactgatc actgatcaat 8100
tattaatcaa tgatcactga taactataaa ctcaaaaaca aaatcatgtg gggattaaga 8160
gaaatgtatc agttttatgt tgtatttctg gtccctgata ctggctcagg taatgccact 8220
attgtcaaga agataccact tgtaaagtag atttaatttt cattatattt taccatatgc 8280
ttctccattc atgacatctc ttgagatgtt gtggtttata ctttcagttt ttctccagtc 8340
catccgcaaa tatcaggcat ctactgtgtt ccaagatatt aaagaaatca tcatgactta 8400
gcctcatcaa cagcattgct agatctggga tggaaaggaa gagtataatcctggcagtca 8460
ggaagaaggc agcataaagt ataagtttct gcttccaaaa aaggtctctc atcagcctgt 8520
agggagtgtg tagggaaggg acagctgtcc ttgtagtagg gaagggtttt attcaggtcg 8580
tctgggctcc ataatatccc ttgtgtatct gcagtctcct ttgccatgga tcaacacaat 8640
aggaaatctt ccggcactga tggtttttcc aagggggagt tcttcctgga gcaaagcaaa 8700
tgaccaacca ggtttgagga cctgatttgt ttgacaattc cattttgtat tgtaaattac 8760
ttaattggca ttctactccc aatccatctt gtcatttgca tacagtggtt ttgggattga 8820
gttcagctat accaaaagtc tgaaccttct gcacttagaa caaggcaacc accaagcttc 8880
acttgcactg aggccgtgtc tccaatggaa atgaggcagc tggcttgcag gagcttccca 8940
actcagggaa gtagaactcc tgagtcacct ccatatgcaa atgatttcac agtaatgctg 9000
ttgaacttca cttcccatca cagcaaatgt gtggtaacat agcttcccca caggagttta 9060
ctcaccatgg tattttaaag gtgaaacatt tcaaaactga aatttgaaag aatttagttt 9120
tggattcact caattatcac tatcacttcg ggtgttattg cacctttctt gtttgtgagt 9180
ttaaatgcca gactctcagg ccactaactt tcaattaaaa gtgtttttct ttaatcgctg 9240
aacctaacag cagggaaaac gaaatgttca ttcagacttt cagaaccttc aatgagatta 9300
ggcagctgaa agatcaaagt gttgcatagt tgtcccgata aagctatttg gatcatatgg 9360
accaaatcga ctgctgtcat tccccaccaa ccccatctct ccccaaaatt cccagccctg 9420
tttaagtgtt ctctgtagca tttatctcta tctagtatat tgtgtagcat atcatatcat 9480
acttttctgt tttgtttatt gtctctctcc tcctagaata taaactccac aagcacaaag 9540
atttgggcct gttttataat attgttgcat ccccagggcc tgatatacag cagagtggtg 9600
gtacgaaaag agcacacaaa aaaatatttg ttgagtcaat gaatgaatga tttcctcaaa 9660
taggattagc ctaaaatttt ggaaacatga acagatttgg atatgtgaaa atttatttcc 9720
agactgttca tcaggaactg ttagcagctt ctaaagggta cactggagca gcagtagtaa 9780
aaggaggaag aggagcagct ctgctactgc tactatcgag tactactaca attagcactt 9840
gcttattctg tgtgttaggc cctgtactga acactctgtc taaattagtt catttcctcc 9900
tggaaatgac tctagggggt aagtgcttca tcatgtaaga tgagtatttt tcacattttg 9960
ttgtgtctga aatctgagtg tgtctttcaa tgatggaatc tttgattcca tgataagtgg 10020
tattattccc attttaagga tgaggaaact gaggtccaaa gaaattaagt aatttgccca 10080
aattcaccca gcctagaaaa tgataaagct agttctaaac ccaagcagat tagctctgaa 10140
gtctgggccc ttaataacca ctttttattg cctatatttg tacctctggt gtacgtatca 10200
agttatatgt tgacttcaaa actatcatga ccttttcttg gttttgattg tccaacatta 10260
gtatagtgtt ctgggtctgc aaaaattttg attactcatc tcatctgtaa aacattttga 10320
actcgtgtgt ttgtgcatgc acatttgtgt gtaattataa aaattttact ttctgttaat 10380
atataagttg tatcataaga aactgccgtt tttgaagagc aaaaaaaggt tgaatgttac 10440
cagttacatc tggttcaacc taatagacat ttgtacaaaa acagacattt taagaggttg 10500
aaataaaaat ttaataaaca atattttcag tttttactaa ttgtgatgct tcactatcat 10560
tagctaatat gtcaaggcat aatatacctt agggtgaact ttatcattaa caaaggtgga 10620
tggtgtcaat aatcttgagg tttgtgtttt tttatataac actgcgaggt ctaattaagt 10680
acttactgtt taccacctca tacagtggcc gataaaaagt gtcacttctg ctgtttcctc 10740
tgggttgtgc ttgaattatt agtattatct tcagtcctca gtttctttgt gggaaacttt 10800
ttaattagtt gtttaatttt gtaagatggt tagtttagtc aaaattagat aagagaattt 10860
gaaaatccgt agctacccca aagcaaccta cacataagaa ctattatttt tgtgttttga 10920
aatcataatt ttattgattt ccagtgtttc cactggtagt ggtttcattg atataggagt 10980
atcaaaacat cactcattat ttatttcagt ttcatttgat cctagccgtt ttgtattaac 11040
tctctgtgaa gaaattacct cacaaatcta ttgctgtcct tggtaaagga atggagaatt 11100
aaggctctag atcattagtg gttacactat agtattagaa gtaaaaaaaa gattatacca 11160
acaaaataag aacatgttaa tgtacttgta atgaataaac atgaataaag ctcttatgct 11220
atataggtgc actaaacaat ctactagaat tgtcagcaaa ctacgtatct taatcctgaa 11280
agggtcccaa accaatgatc taaaattgaa tcaaactttc ttccttgagc ataattactt 11340
aaatgattta ttaaaatagc cagcatttaa aagcttaaaa tgtaaatatc ataatgtggt 11400
atcctagata gcatcccaga acagaaaaag gatattaggg aaaaactgga ggaatggaat 11460
aaattatgca gtttagttat taataatgta ctaacgtcct tagttatgac gattgtacca 11520
tggtaatgta agatactaac aatagaggaa accgggtaag gagtatacag taactctata 11580
ctatctttgc aacttttttg taaatttaaa acttctaaaa taaagaacaa atttaaacat 11640
taaaaagtat caccaggaac atatatcact gtttacagat gaaatactat gtattttcat 11700
atctaatttc tgatcattga cttcaaatca gaaaagtgaa tgacacctca aaatcaggtt 11760
ttctgtttac tgaagtctaa gaaaagaaag cataccagct ggagagattc atgtttataa 11820
agacagattt ataacaacaa aaataaaata tccaagaata aatttaagaa gaagcacttt 11880
actgagaaac atatgaaaac ctgaacaaat ggagagggat attttgtatt tgaatagaaa 11940
gacttctggt ttaaagataa ttctctttaa attatttttt gtagaaattt aaggggtaca 12000
agagcagtgt tgtcacatgg atatattaca tagtggtgaa gtctggggtt ttagtgtaaa 12060
ttaatcttta cattttgttt gagcccaata aatgtaccaa catgattttt atagaaagat 12120
agtcattcct attaatccaa acttgtccca actttgaatt gaattgaggc agagctagca 12180
ggtgttcccc acggctgagg catctgaaca ttaagcatat ccctctgaga accagcctgc 12240
attgatactc tttctaatgt ggacagcatc aagctatgta cgtagttctg tgctcagcaa 12300
aagccctgac ttctttttgt ttatgtccta gccccataca acaaaatcaa ccaaagaatt 12360
ttggttgtgg atccagtcac ctctgaacat gaactgacat gtcaggctga gggctacccc 12420
aaggccgaag tcatctggac aagcagtgac catcaagtcc tgagtggtaa gaccaccacc 12480
accaattcca agagagagga gaagcttttc aatgtgacca gcacactgag aatcaacaca 12540
acaactaatg agattttcta ctgcactttt aggagattag atcctgagga aaaccataca 12600
gctgaattgg tcatcccagg taatattctg aatgtgtcca ttaaaatatg tctaacactg 12660
tcccctagca cctagcatga tgtctgccta tcatagtcat tcagtgattg ttgaataaat 12720
gaatgaatga ataacactat gtttacaaaa tatatcctaa ttcctcacct ccattcatcc 12780
aaaccatatt gttacttaat aaacattcag cagatattta tggaatatac cttttgttcc 12840
atgcattgta gtactcattg gatacacata gaataataag actcagttca cactcttcag 12900
gaaacagata aaaaactaag aaacaaacaa aaaacaggca atccaacacc atgtgggaaa 12960
tgctttcata gccgggaaac ctggggaata cctgagagga atactcaatt caggccttgt 13020
ttcaggaatc caaatcctgg cacatcagag ctgcttccct ctttccaggg tggcaggaaa 13080
taaatggaac atatttttct atcttatgcc aaacatgagg gaccctttct ccccggtgcc 13140
tctcccaagg tagtctacaa tatttcaact ctagcagtct gcttagtgca tagaacatga 13200
ggctgtgtgt ccctgggcaa attactagac ttctgtgtgc ttcactttcc ctgtaggatt 13260
ataatctact gagcaagctt attgtaaggg tcagattagc aacagtgtat gaaaatgatt 13320
tgagaccatt gcctgcacaa attcaactat ttttttttat ctcactactc tacagaagta 13380
ggtagggtgg gagacagagt ctgatgagag gctcagaatg tgaaagaaag tgaggcgagt 13440
gagcatgata tttaatataa acacaaagat attctgagaa gagctgctca ctgccccctc 13500
ccccaataca tgttgatagg aaaatgccac gtacttcagc aaaaacaact gaaaaattag 13560
atagaaaagt caatcaatag gaaaagataa tccaggacgg tgttgtgaac agaaagaggg 13620
ggaaaaaact ttagaaaatg atggggatgc tcttactggg gtacgagtcc tcaggtattg 13680
aactggcttt cagtaaaagc tagattagtg ggttcctgcc atttacaagc tgttttatga 13740
caacttactt gttgggtggc ctacagtaac tcacctaact gcactgagtc tgtttcctca 13800
tctgtaaatt ggggattttt ttttaaatac ctggcatgcc taactcataa agttgttctg 13860
aaactgaaat aaaacatacg tgaacaggca ttgtaaactg taagttacgg aaaaagctgg 13920
ctgttgttgt gtctttaaag tttcacctgg gtagtcaaag atggatcatg ggtctcagtg 13980
gagagctgag ccaggcagga gctgactaag ggtgagaggt gggagttagc agcctctgaa 14040
catctgtgta ccatgggacc ccctttcctc ctgcatggta ccccagacaa ggagcctagt 14100
aagagatact aatggcttgt tgtccagaga tgttcaaact gcagagaaag ataagacaac 14160
aagcattggc ctccaatcat gatgacagat aggaggaggt gggagctcct tagcagtgct 14220
ggttggcctt ccatgttcta ctgtgggcca tctctgccat gtactgtagg ctactagctt 14280
ctatattaaa gaatgcaaga ggggccagga gcggaggctc atgcctgtaa tctcagcact 14340
ttgggaggcc aaggtgggca gatcacttga ggtcaggagt ttgtgaccag cctggccaac 14400
atggtgaaac tctgccttta ctaaaaatat aaaaattagc tgggtgtggt ggtgtgcacc 14460
tgtaatccca gctactcggg agactgaggc acaagaattg cttgaacctg ggaggcggaa 14520
gttgcagtga gcccagattg cgccactgca ctccaccctg ggcaacagag aaagactctg 14580
cctcaaaaaa aaaaaaaaaa agcaagagga agtgaaataa tcaaggccgc catttaatag 14640
tgagcagcca ctccatgtgg tactgtgcaa gcacattata aatattagcc tcacaagaaa 14700
tgtattagca tttgtatttt gtacactggt taagtatctt gcccaagacc tcaaaactgg 14760
ttaagggcag cagaatttag ccccagcacc accttttcaa agcctgggct tctcacactt 14820
ctccatgctg ttcccatttt aacacaggta tctcgccatt ccagccactc aaactttggc 14880
atttaagaaa attatcctaa agctaaacta aacttcaagg atgaccattc tcctgacccc 14940
ttcccatcaa aattttatct ttagtcagtt tgttttcgtt ttgttttgtt tttcagaact 15000
acctctggca catcctccaa atgaaaggac tcacttggta attctgggag ccatcttatt 15060
atgccttggt gtagcactga cattcatctt ccgtttaaga aaaggtagta tttccttaat 15120
tgcagtggtc tccactgggg gtgaggaagg ggtgagaatt ggatcatggc tgcaaggaaa 15180
cccgacttaa cctctgcaag gtggtgcaaa ggcattccac tgttcaacag caattatatt 15240
gaagctgagt gggatcactg ggtgaagatg aagcgtaagg ggtgaggggc aggagaatgg 15300
gtatggatgg aggtagaaga tgcagtgtca tacagttttt ttctatcatg aaaataacca 15360
cagacttaca gaagagaaag agctaaaatg cccgtcattt tcagttgcat tttagtcttg 15420
cattagttgc aaccagctgg tttctgggta ccctaagtaa taaaaatagt tcctctgtag 15480
aactgtagta tgtttaccat agagtatttt gcaaaatttt tggtagagga tgttacataa 15540
tttgcatgtg ttcatttctc catttacctg tgggaacaat taaaatccag gaaaatgagt 15600
atattcaaat aatttcctcc catttaagat gagtcagagt aaataattcc tccaatactt 15660
agagaagtat accaagagat ccagtgatgg tatagagttg tctgatgtta aatagggaag 15720
tagaatatgg aaggggattc caatagtcgt tgaaaaattc cccataaccc cttacatggg 15780
ggaaagtagt gttaactgag agagtagaga taagctgttt ccaaaaatta tattcttaac 15840
aggactgaga tagccagaat ataaggatca agtttcaatg acagtaagat cctgagatgg 15900
agttgatttg cacaaagaaa taattgttgc cagcatgcat tttgaatatt tctctggaaa 15960
aaaagattag ttggcagtag aaatggatag aaatcaatag atattaaaat acctcagaat 16020
ttggttcatc tctgggaaaa gatgaaaaat aaaagtgtat actcctcaag aacatctagg 16080
atcaaaagca tgtgccctac actattgaat taattaacct cataagttgg gacctgtgga 16140
ataaggatgt ccaccagact tcctagggat tacaaatgtt tcacagaact tgaaatttaa 16200
acttgggtca ctgtatggga tgtagagctg tgctatatgg aaataaaaat gatttctttt 16260
tctcaaggga gaatgatgga tgtgaaaaaa tgtggcatcc aagatacaaa ctcaaagaag 16320
caaagtggta agaatatcag aaggaattgg gaagtaaaag tcaaaggaaa caaaaagcta 16380
aagcaataac aaagagaaat ccatcagtca taatctcctc tccttttaaa gaatgctggt 16440
tcccctttgc ctcacagcta acacaagaac tcctccaccg tctgaggagg tttaggagca 16500
gggaagggga aggagtcagc ttcatttgct aatcttctgt tgccctgcac cctagcagct 16560
ccttgcagca ggggacaagg atgacttagg tggatggata attaattgat tctaaaatat 16620
tgtgtgtcag tattgtaata ctatgttaat tgcaccatgc acggtatctc atttaatccc 16680
ccaccccttg ccattaccaa agagagagag agagagagag agagaaatac tagaatttat 16740
cctcatttta cagtagagaa aacagagggt caagaagata atgtaaagtg cccaagaaca 16800
cacagctgat cacaaaaatc aagcttgggg gccattagcc taaccacaga cccttactct 16860
taacccatct gcttcaatcc attttgctac aaatgtttac atttataagc agggcagaaa 16920
aacctcatcc aggttattga actaagaaga aagttatatt aaggtttcta atttttttaa 16980
tgtagttaga aaccaaactt aacaatgagc ccaagtttaa agcagtctaa ttaacctgga 17040
caagctcagg caagtttcat tctgtggccc atagcatcat ctgtgttgta aagctaagta 17100
gcaaatgttg tttgggtcat gctgggggac aagccatccc aatttgctca ggactgaggg 17160
gttttccagg atatcatgta aggataattg ggtacaaata taacctgctg ctttctctca 17220
tttcaaattt atcatttatc atatcagcaa ctatgagtta tgttttttat tagatttctt 17280
gttacttttt ccccagacca cttcccatga aattaatata ctattatcac tctccagata 17340
cacatttgga ggagacgtaa tccagcattg gaacttctga tcttcaagca gggattctca 17400
acctgtggtt taggggttca tcggggctga gcgtgacaag aggaaggaat gggcccgtgg 17460
gatgcaggca atgtgggact taaaaggccc aagcactgaa aatggaacct ggcgaaagca 17520
gaggaggaga atgaagaaag atggagtcaa acagggagcc tggagggaga ccttgatact 17580
ttcaaatgcc tgaggggctc atcgacgcct gtgacaggga gaaaggatac ttctgaacaa 17640
ggagcctcca agcaaatcat ccattgctca tcctaggaag acgggttgag aatccctaat 17700
ttgagggtca gttcctgcag aagtgccctt tgcctccact caatgcctca atttgttttc 17760
tgcatgactg agagtctcag tgttggaacg ggacagtatt tatgtatgag tttttcctat 17820
ttattttgag tctgtgaggt cttcttgtca tgtgagtgtg gttgtgaatg atttcttttg 17880
aagatatatt gtagtagatg ttacaatttt gtcgccaaac taaacttgct gcttaatgat 17940
ttgctcacat ctagtaaaac atggagtatt tgtaaggtgc ttggtctcct ctataactac 18000
aagtatacat tggaagcata aagatcaaac cgttggttgc ataggatgtc acctttattt 18060
aacccattaa tactctggtt gacctaatct tattctcaga cctcaagtgt ctgtgcagta 18120
tctgttccat ttaaatatca gctttacaat tatgtggtagcctacacaca taatctcatt 18180
tcatcgctgt aaccaccctg ttgtgataac cactattatt ttacccatcg tacagctgag 18240
gaagcaaaca gattaagtaa cttgcccaaa ccagtaaata gcagacctca gactgccacc 18300
cactgtcctt ttataataca atttacagct atattttact ttaagcaatt cttttattca 18360
aaaaccattt attaagtgcc cttgcaatat caatcgctgt gccaggcatt gaatctacag 18420
atgtgagcaa gacaaagtac ctgtcctcaa ggagctcata gtataatgag gagattaaca 18480
agaaaatgta ttattacaat ttagtccagt gtcatagcat aaggatgatg cgaggggaaa 18540
acccgagcag tgttgccaag aggaggaaat aggccaatgt ggtctgggac ggttggatat 18600
acttaaacat cttaataatc agagtaattt tcatttacaa agagaggtcg gtacttaaaa 18660
taaccctgaa aaataacact ggaattcctt ttctagcatt atatttattc ctgatttgcc 18720
tttgccatat aatctaatgc ttgtttatat agtgtctggt attgtttaac agttctgtct 18780
tttctattta aatgccacta aattttaaat tcataccttt ccatgattca aaattcaaaa 18840
gatcccatgg gagatggttg gaaaatctcc acttcatcct ccaagccatt caagtttcct 18900
ttccagaagc aactgctact gcctttcatt catatgttct tctaaagata gtctacattt 18960
ggaaatgtat gttaaaagca cgtattttta aaattttttt cctaaatagt aacacattgt 19020
atgtctgctg tgtactttgc tatttttatt tattttagtg tttcttatat agcagatgga 19080
atgaatttga agttcccagg gctgaggatc catgccttct ttgtttctaa gttatctttc 19140
ccatagcttt tcattatctt tcatatgatc cagtatatgt taaatatgtc ctacatatac 19200
atttagacaa ccaccatttg ttaagtattt gctctaggac agagtttgga tttgtttatg 19260
tttgctcaaa aggagaccca tgggctctcc agggtgcact gagtcaatct agtcctaaaa 19320
agcaatctta ttattaactc tgtatgacag aatcatgtct ggaacttttg ttttctgctt 19380
tctgtcaagt ataaacttca ctttgatgct gtacttgcaa aatcacattt tctttctgga 19440
aattccggca gtgtaccttg actgctagct accctgtgcc agaaaagcct cattcgttgt 19500
gcttgaaccc ttgaatgcca ccagctgtca tcactacaca gccctcctaa gaggcttcct 19560
ggaggtttcg agattcagat gccctgggag atcccagagt ttcctttccc tcttggccat 19620
attctggtgt caatgacaag gagtaccttg gctttgccac atgtcaaggc tgaagaaaca 19680
gtgtctccaa cagagctcct tgtgttatct gtttgtacat gtgcatttgt acagtaattg 19740
gtgtgacagt gttctttgtg tgaattacag gcaagaattg tggctgagca aggcacatag 19800
tctactcagt ctattcctaa gtcctaactc ctccttgtgg tgttggattt gtaaggcact 19860
ttatcccttt tgtctcatgt ttcatcgtaa atggcatagg cagagatgat acctaattct 19920
gcatttgatt gtcacttttt gtacctgcat taatttaata aaatattctt atttattttg 19980
ttacttggta caccagcatg tccattttct tgtttatttt gtgtttaata aaatgttcag 20040
tttaacatcc cagtggagaa agtta 20065
Claims (17)
1. A method of treating natural killer cell/T cell lymphoma in a subject, comprising administering to the subject a therapeutically effective amount of pembrolizumab, wherein the subject is characterized by the presence of at least one JAK3 activation mutation or at least one PD-L1 structural rearrangement.
2. A method of determining the response of a subject having a natural killer/T-cell lymphoma to pembrolizumab treatment, the method comprising:
-obtaining a sample from the subject;
-detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement;
wherein the presence of the at least one JAK activating mutation or at least one PD-L1 structural rearrangement indicates that the subject will respond to pembrolizumab therapy.
3. The method of claim 3, wherein the subject is administered a compound that blocks the PD-1/PD-L1 axis.
4. The method of claim 3, wherein the compound is selected from the group consisting of: nivolumab (opdivo), pembrolizumab (keytruruda), alemtuzumab (tecentriq), avizumab (bavencio), coviruzumab (imfinzi), piduzumab, AMP-224, AMP-514, PDR001, cimiciprizumab, and combinations thereof.
5. The method according to any preceding claim, wherein the JAK3 activating mutation is selected from the group consisting of M511I, A572V, A573V, R657Q, V722I, V674A, L857P, R403H, Q501H, E958K, and combinations thereof.
6. The method of claim 5, wherein the JAK3 activating mutation is A572V.
7. The method of any one of the preceding claims, wherein the PD-L1 structural rearrangement is a mutation in the PD-L1 gene.
8. The method of claim 7, wherein the mutation is selected from the group consisting of: insertions, deletions, substitutions, translocations, inversions, micro-inversions, duplications, tandem repeats, breakpoints (mutations), and combinations thereof.
9. The method of any one of claims 7-8, wherein the mutation in the PD-L1 gene disrupts the 3' UTR of the PD-L1 gene.
10. A method of treating a natural killer/T cell lymphoma in a subject comprising administering to the subject an inhibitor selected from the group consisting of a PD-1 inhibitor, a CD279 inhibitor, a PD-L1 inhibitor, a CD274 inhibitor, and combinations thereof.
11. The method of claim 10, further comprising administering pembrolizumab.
12. The method of any one of the preceding claims, wherein the natural killer/T-cell lymphoma is extranodal natural killer/T-cell lymphoma.
13. The method of any one of the preceding claims, wherein the natural killer/T-cell lymphoma is relapsed and/or refractory natural killer/T-cell lymphoma.
14. A kit for detecting the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement comprising a detection agent and at least one pair of primers, wherein the primers are enriched for the genomic region of the JAK3 and PD-L1 genes.
15. The kit of claim 14, wherein the at least one pair of primers is selected from the group consisting of: SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26, SEQ ID NO: 27 and 28, SEQ ID NO: 29 and 30, SEQ ID NO: 31 and 32, SEQ ID NO: 33 and 34, SEQ ID NO: 35 and 36, SEQ ID NO: 37 and 38, SEQ ID NO: 39 and 40, SEQ ID NO: 41 and 42, SEQ ID NO: 43 and 44, SEQ ID NO: 45 and 46, and SEQ ID NO: 47 and 48.
16. A kit for next generation sequencing that detects the presence or absence of at least one JAK3 activating mutation or at least one PD-L1 structural rearrangement.
17. The kit according to any one of claims 14 to 16 for use in a method according to any one of claims 2 to 13.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SG10201708262R | 2017-10-06 | ||
| SG10201708262R | 2017-10-06 | ||
| PCT/SG2018/050509 WO2019070204A1 (en) | 2017-10-06 | 2018-10-08 | Methods for treating lymphomas |
Publications (1)
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|---|---|
| CN111479932A true CN111479932A (en) | 2020-07-31 |
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| CN201880078523.4A Pending CN111479932A (en) | 2017-10-06 | 2018-10-08 | Method of treating lymphoma |
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| US (1) | US20200325229A1 (en) |
| EP (1) | EP3692174A4 (en) |
| JP (1) | JP2020536111A (en) |
| CN (1) | CN111479932A (en) |
| SG (1) | SG11202003127WA (en) |
| WO (1) | WO2019070204A1 (en) |
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| WO2022241293A2 (en) * | 2021-05-14 | 2022-11-17 | Foundation Medicine, Inc. | Cd274 mutations for cancer treatment |
| CN114507286B (en) * | 2022-04-18 | 2022-07-05 | 苏州百道医疗科技有限公司 | anti-PD-L1 recombinant rabbit monoclonal antibody and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104220051A (en) * | 2011-11-25 | 2014-12-17 | 新加坡保健服务集团有限公司 | Natural-killer/T-cell lymphoma (NKTCL) susceptibility prediction, diagnosis and therapy |
| US20170115291A1 (en) * | 2014-05-28 | 2017-04-27 | Dana-Farber Cancer Institute, Inc. | Activating JAK Kinase Biomarkers Predictive of Anti-Immune Checkpoint Inhibitor Response |
| CN106987631A (en) * | 2017-04-01 | 2017-07-28 | 武汉赛云博生物科技有限公司 | A kind of immune group sequencing technologies for the adjoint diagnosis of PD 1/PD L1 blocking treatments |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016175275A1 (en) * | 2015-04-30 | 2016-11-03 | 国立大学法人京都大学 | Method for predicting therapeutic effect of pd-1/pd-l1 inhibitor using abnormality in pd-l1(cd274) as index |
-
2018
- 2018-10-08 EP EP18863900.9A patent/EP3692174A4/en not_active Withdrawn
- 2018-10-08 JP JP2020519426A patent/JP2020536111A/en active Pending
- 2018-10-08 SG SG11202003127WA patent/SG11202003127WA/en unknown
- 2018-10-08 CN CN201880078523.4A patent/CN111479932A/en active Pending
- 2018-10-08 WO PCT/SG2018/050509 patent/WO2019070204A1/en unknown
- 2018-10-10 US US16/754,068 patent/US20200325229A1/en not_active Abandoned
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104220051A (en) * | 2011-11-25 | 2014-12-17 | 新加坡保健服务集团有限公司 | Natural-killer/T-cell lymphoma (NKTCL) susceptibility prediction, diagnosis and therapy |
| US20170115291A1 (en) * | 2014-05-28 | 2017-04-27 | Dana-Farber Cancer Institute, Inc. | Activating JAK Kinase Biomarkers Predictive of Anti-Immune Checkpoint Inhibitor Response |
| CN106987631A (en) * | 2017-04-01 | 2017-07-28 | 武汉赛云博生物科技有限公司 | A kind of immune group sequencing technologies for the adjoint diagnosis of PD 1/PD L1 blocking treatments |
Non-Patent Citations (4)
| Title |
|---|
| JIANPING LAI等: "Successful treatment with antiprogrammed-death-1 antibody in a relapsed natural killer/T-cell lymphoma patient with multi-line resistance: a case report", 《BMC CANCER》 * |
| KEISUKE KATAOKA等: "Aberrant PD-L1 expression through 3′-UTR disruption in multiple cancers", 《NATURE》 * |
| SUNG HOON SIM等: "Novel JAK3-Activating Mutations in Extranodal NK/T-Cell Lymphoma, Nasal Type", 《THE AMERICAN JOURNAL OF PATHOLOGY》 * |
| YOK-LAM KWONG等: "PD1 blockade with pembrolizumab is highly effective in relapsed or refractory NK/T-cell lymphoma failing L-asparaginase", 《BLOOD》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| US20200325229A1 (en) | 2020-10-15 |
| SG11202003127WA (en) | 2020-05-28 |
| EP3692174A4 (en) | 2021-06-23 |
| EP3692174A1 (en) | 2020-08-12 |
| JP2020536111A (en) | 2020-12-10 |
| WO2019070204A1 (en) | 2019-04-11 |
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